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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