Bone marrow-derived stromal cells (BMSCs) protect against acute lung injury (ALI). To determine the role of BMSC mitochondria in the protection, we airway-instilled mice first with lipopolysaccharide (LPS), then with mouse BMSCs (mBMSCs). Live optical studies revealed that mBMSCs formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the alveolar epithelium, releasing mitochondria-containing microvesicles that the epithelium engulfed. The presence of BMSC mitochondria in the epithelium was evident optically, as also by the presence of human mitochondrial DNA in mouse lungs in which we instilled human BMSCs (hBMSCs). The mitochondrial transfer increased alveolar ATP. LPS-induced ALI, indicated by alveolar leukocytosis and protein leak, inhibition of surfactant secretion and high mortality, was markedly abrogated by wild type mBMSCs, but not by mutant, GJC-incompetent mBMSCs, or by mBMSCs with dysfunctional mitochondria. This is the first evidence that BMSCs protect against ALI by restituting alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer.

Mesenchymal stem cells (MSCs), which potentially transdifferentiate into multiple cell types, are increasingly reported to be beneficial in models of organ system injury. However, the molecular mechanisms underlying interactions between MSCs and host cells, in particular endothelial cells (ECs), remain unclear. We show here in a matrigel angiogenesis assay that MSCs are capable of inhibiting capillary growth. After addition of MSCs to EC-derived capillaries in matrigel at EC: MSC ratio of 1:1, MSCs migrated toward the capillaries, intercalated between ECs, established Cx43-based intercellular gap junctional communication (GJC) with ECs, and increased production of reactive oxygen species (ROS). These events led to EC apoptosis and capillary degeneration. In an in vivo tumor model, direct MSC inoculation into subcutaneous melanomas induced apoptosis and abrogated tumor growth. Thus, our findings show for the first time that at high numbers, MSCs are potentially cytotoxic and that when injected locally in tumor tissue they might be effective antiangiogenesis agents suitable for cancer therapy. IntroductionIntense interest in the therapeutic application of bone marrowderived mesenchymal stem cells (MSCs) arises from the possibility that MSCs promote vascular repair. In animal models, intravenous injections of MSCs protected against heart failure by enhancing cardiac myocyte survival 1 and blocked lipopolysaccharide-induced acute lung injury by reducing total cell and proinflammatory cytokines in the lung. 2 In a collagen gel model, MSCs promoted survival of capillaries grown from human umbilical vein endothelial cells (HUVECs). 3 Despite these findings, the lack of conclusive evidence supporting a beneficial effect of MSCs in the clinical setting 4 indicates that mechanisms underlying MSC-endothelial cell (EC) interactions require better understanding.Several reports indicate that these interactions result from direct contact between MSCs and host cells. The MSC-induced responses include induction of gene transcription in ECs, 3 mitochondrial transfer in A549 cells, 5 and interleukin-10 (IL-10) secretion in alveolar macrophages. 6 In the context of tumor growth, MSCs recruit ECs to induce angiogenesis in stable tissue 7 as well as in tumors, 8 raising the possibility that MSCs might promote tumor growth. By contrast, intravenously injected MSCs are capable of abrogating growth of the Kaposi sarcoma, 9 suggesting that MSCs potentially possess cytotoxic properties. However, the mechanisms by which MSCs engage ECs are not understood and might involve gap junctional communication (GJC), as proposed for MSCcardiomycyte interactions. 10 Here, we addressed MSC-EC interactions in a capillary culture with the expectation that MSCs would enhance angiogenesis. However, surprisingly, addition of MSCs caused dose-dependent EC cytotoxicity that was attributable to the formation of MSC-EC GJC and the production of MSC-derived reactive oxygen species (ROS). The combined effect of these responses was capillary destruction. Further...

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.

The European Respiratory Society (ERS) Research Seminar entitled "Pulmonary vascular endothelium: orchestra conductor in respiratory diseases - highlights from basic research to therapy" brought together international experts in dysfunctional pulmonary endothelium, from basic science to translational medicine, to discuss several important aspects in acute and chronic lung diseases. This review will briefly sum up the different topics of discussion from this meeting which was held in Paris, France on October 27-28, 2016. It is important to consider that this paper does not address all aspects of endothelial dysfunction but focuses on specific themes such as: 1) the complex role of the pulmonary endothelium in orchestrating the host response in both health and disease (acute lung injury, chronic obstructive pulmonary disease, high-altitude pulmonary oedema and pulmonary hypertension); and 2) the potential value of dysfunctional pulmonary endothelium as a target for innovative therapies.

Shedding of the extracellular domain of cytokine receptors allows the diffusion of soluble receptors into the extracellular space; these then bind and neutralize their cytokine ligands, thus dampening inflammatory responses. The molecular mechanisms that control this process, and the extent to which shedding regulates cytokine-induced microvascular inflammation, are not well defined. Here, we used real-time confocal microscopy of mouse lung microvascular endothelium to demonstrate that mitochondria are key regulators of this process. The proinflammatory cytokine soluble TNF-α (sTNF-α) increased mitochondrial Ca 2+ , and the purinergic receptor P 2 Y 2 prolonged the response. Concomitantly, the proinflammatory receptor TNF-α receptor-1 (TNFR1) was shed from the endothelial surface. Inhibiting the mitochondrial Ca 2+ increase blocked the shedding and augmented inflammation, as denoted by increases in endothelial expression of the leukocyte adhesion receptor E-selectin and in microvascular leukocyte recruitment. The shedding was also blocked in microvessels after knockdown of a complex III component and after mitochondria-targeted catalase overexpression. Endothelial deletion of the TNF-α converting enzyme (TACE) prevented the TNF-α receptor shedding response, which suggests that exposure of microvascular endothelium to sTNF-α induced a Ca 2+ -dependent increase of mitochondrial H 2 O 2 that caused TNFR1 shedding through TACE activation. These findings provide what we believe to be the first evidence that endothelial mitochondria regulate TNFR1 shedding and thereby determine the severity of sTNF-α-induced microvascular inflammation.