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...

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.

The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na ؉ -dependent and highly selective. The K m for tryptophan was ϳ145 nM, and tryptophan transport was unchanged in the presence of 100 M concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K I values of 200 and 440 M, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His 6 tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.Transporters responsible for reuptake of neurotransmitters across the plasma membrane of neurons and glia fall into two gene families (1). The majority of small neurotransmitters, including glycine, ␥-aminobutyric acid (GABA), 1 dopamine, norepinephrine, and 5-hydroxytryptamine (5-HT, serotonin), are transported by proteins belonging to the family designated the neurotransmitter:sodium symporter (NSS) family 2.A.22 by Saier (2). Glutamate, however, is transported by a family of mono-and dicarboxylic amino acid transporters, the dicarboxylate/amino acid:cation symporters family (2). Proteins in both families play important roles in brain function as indicated by the profound behavioral effects of drugs that influence their activity, such as cocaine and amphetamines, which interact with amine transporters in the NSS family (3-12), and many antidepressant drugs that inhibit serotonin and norepinephrine transporters (13-17).Among the sequences found to be homologous to the NSS family of transporters are a number of "orphan" transporters, for which no function is known. These orphans include v7-3 (18), NTT4 (19,20), inebriated (21), blot (22), and NTT5 (23), among others. The largest number of orphan sequences in this family is found in prokaryotic organisms. Although these orphan sequences are highly similar to those encoding functional transporters, it is possible that these proteins fulfill other functions. For example, within the ATP-binding cassette family of transporters are the sulfonylurea receptor (24) and the cystic fibrosis transmembrane regulator chloride channel (25). In the dicarboxylate/amino acid:cation symporters neurotransmitter transporter family is EAAT4, a ligand-gated ion channel (26); SGLT3, a member of the sodium:solute symporter (SSS) sugar transporter family, also is not a transporter but rather a glucose-gated ion channel.2 Moreover, some proteins, such as adenylate cyclase (28) and patched (29) also have 12 transmembrane segments but no known transport function. For the orphan transporters in the NSS family, it is important to know if any of the newly discovered prokaryotic sequences actually encode functional transporters.Symbiobacterium thermophilum is a symbiotic thermophile, the growth of which is dependent on co-culture with an associated...

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Alveolar type 2 (AT2) cells secrete surfactant that forms a protective layer on the lung's alveolar epithelium. Vesicles called lamellar bodies (LBs) store surfactant. Failure of surfactant secretion, which causes severe lung disease, relates to the manner in which LBs undergo exocytosis during the secretion. However, the dynamics of LBs during the secretion process are not known in intact alveoli. Here, we addressed this question through real-time confocal microscopy of single AT2 cells in live alveoli of mouse lungs. Using a combination of phospholipid and aqueous fluorophores that localize to LBs, we induced surfactant secretion by transiently hyperinflating the lung, and we quantified the secretion in terms of loss of bulk LB fluorescence. In addition, we quantified inter-LB phospholipid flow through determinations of fluorescence recovery after photobleaching. Furthermore, we determined the role of F-actin in surfactant secretion through expression of the fluorescent F-actin probe Lifeact. Our findings indicate that, in AT2 cells in situ, LBs are held in an F-actin scaffold. Although F-actin transiently decreases during surfactant secretion, the LBs remain stationary, forming a chain of vesicles connected by intervesicular channels that convey surfactant to the secretion site on the plasma membrane. This is the first instance of a secretory process in which the secretory vesicles are immobile, but form a conduit for the secretory material.

Although exposure to ambient hypoxia is known to cause proinflammatory vascular responses, the mechanisms initiating these responses are not understood. We tested the hypothesis that in systemic hypoxia, erythrocyte-derived H 2 O 2 induces proinflammatory gene transcription in vascular endothelium. We exposed mice or isolated, perfused murine lungs to 4 hours of hypoxia (8% O 2 ). Leukocyte counts increased in the bronchoalveolar lavage. The expression of leukocyte adhesion receptors, reactive oxygen species, and protein tyrosine phosphorylation increased in freshly recovered lung endothelial cells (FLECs). These effects were inhibited by extracellular catalase and by the removal of erythrocytes, indicating that the responses were attributable to erythrocyte-derived H 2 O 2 . Concomitant nuclear translocation of the p65 subunit of NF-kB and hypoxiainducible factor-1a stabilization in FLECs occurred only in the presence of erythrocytes. Hemoglobin binding to the erythrocyte membrane protein, band 3, induced the release of H 2 O 2 from erythrocytes and the p65 translocation in FLECs. These data indicate for the first time, to our knowledge, that erythrocytes are responsible for endothelial transcriptional responses in hypoxia.Keywords: hypoxia; erythrocytes; endothelium; lung; inflammation Systemic hypoxia, which is characterized by a decrease in the partial pressure of oxygen in blood (PO 2 ), results from a lack of oxygen in inhaled breath, or from impaired blood oxygenation because of lung disease. The circulatory response to a decrease of PO 2 is best characterized by pulmonary arterial vasoconstriction, a protective strategy that redistributes the pulmonary blood flow from hypoxic to well-ventilated regions of the lung. An additional vascular effect may involve a hypoxia-induced innate immune response, characterized by leukocyte activation and tissue injury (1). However, this effect remains controversial. Support for the immune effect derives from evidence in animal models that ambient hypoxia causes lung injury (2-5), systemic inflammation (4, 5), and vascular leakage (2, 6). A 4-hour exposure to 8% O 2 in mice activates NF-kB in astrocytes and hepatocytes (7), suggesting that hypoxia-induced proinflammatory gene transcription occurs in these cells.The evidence opposing the hypoxic immune response comes from lung lymph flow studies in adult sheep. According to these studies, ambient hypoxia does not increase lung microvascular permeability to proteins, and it does not cause pulmonary edema (8, 9). The clinical evidence for the hypoxic immune effect is mixed, and is based on studies of high-altitude pulmonary edema (HAPE), a form of lung injury that follows exposure to hypoxia at high altitude. Radioactive tracer studies in patients with HAPE confirm the sheep data insofar as lung microvascular permeability does not increase (10). Several studies indicate that the bronchoalveolar lavage (BAL) of patients with HAPE is enriched in leukocytes, proteins, and proinflammatory factors, indicating that HAPE causes lu...

Although the lung expresses procoagulant proteins under inflammatory conditions, underlying mechanisms remain unclear. Here, we addressed lung endothelial expression of tissue factor (TF), which initiates the coagulation cascade and expression of which signifies development of a procoagulant phenotype in the vasculature. To establish the model of acid-induced acute lung injury (ALI), we intranasally instilled anesthetized mice with saline or acid. Then 2 h later, we isolated pulmonary vascular cells for flow cytometry and confocal microscopy to detect the leukocyte antigen, CD45 and the endothelial markers VE-cadherin and von Willebrand factor (vWf). Acid increased both the number of vWf-expressing cells as well as TF and P-selectin expressions on these cells. All of these effects were markedly inhibited by treating mice with antiplatelet serum, suggesting the involvement of platelets. The increased expressions of TF, vWf, and P-selectin in response to acid also occurred in platelets. Moreover, the effects were replicated in endothelial cells derived from isolated, blood-perfused lungs. However, the effect was inhibited completely in lungs perfused with platelet-depleted and, to a lesser extent, with leukocyte-depleted blood. Acid injury increased endothelial expressions of the platelet proteins, CD41 and CD42b, providing evidence that platelet proteins were transferred to the vascular surface. Reactive oxygen species (ROS) were implicated in these responses, in that the endothelial and platelet protein expressions were inhibited. We conclude that acid-induced ALI causes NOX2-mediated ROS generation that activates platelets, which then generate a procoagulant endothelial surface.