Our findings indicate that alveolar attachment, hence lung retention of the migration-prone BMSCs was critical for the ensuing protective effects against ALI. Following alveolar attachment, BMSCs transferred mitochondria to the alveolar epithelium, an event that occurred progressively for 24 h, accounting for the protective effect. BMSCs carrying defective mitochondria failed to protect. Cx43 was the critical mechanism underlying alveolar attachment of wild-type BMSCs that generated Cx43-expressing nanotubes and microvesicles in a Ca2+
dependent manner. Epithelial engulfment of the released microvesicles might have contributed to the mitochondrial transfer. Microvesicles formed extensively in the lung and their parenchymal dispersal might have been convective24,37
. Together, these findings provide the first in vivo
evidence that BMSCs establish epithelial interactions, form microstructures and transfer mitochondria to host cells.
The mitochondrial transfer increased alveolar ATP in LPS lungs in a Cx43-dependent manner. Single-cell ATP determinations, the first in an intact organ, indicated that the ATP increase occurred at the transfer site as well as in adjoining alveoli, affirming that the transferred mitochondria were live and capable of ATP generation in the recipient epithelium. Inflation-induced alveolar surfactant secretion, a major ATP-dependent process that regulates alveolar stability38–39
, provides a functional metric of alveolar bioenergetics. The fact that we could induce the secretion in LPS lungs only after instillation of wild-type BMSCs, but not of BMSCs carrying dysfunctional mitochondria, underlines the importance of bioenergetics restoration in cell repair therapy.
We confirmed that BMSCs increased mouse survival in LPS-ALI11
, and we showed further that the survival advantage was lost if the BMSCs contained dysfunctional mitochondria or GJCincompetent Cx43, or if they were depleted of Cx43. These findings further support Cx43-dependent mechanisms and transfer of viable mitochondria in the protective response. Although long-term consequences of mitochondrial transfer need to be understood, evidently the transfer was sufficiently rapid to be protective in the acute phase of lung injury.
Unanswered questions include the relative roles of paracrine secretion11–12,16
versus mitochondrial transfer in the BMSC protective effect. Although mitochondrial loss in alveolus-attached BMSCs could decrease ATP in these cells, thereby decreasing their secretory capacity, protective paracrine secretions could continue from non-attached, mitochondria-competent BMSCs. The combined effects of these different sets of BMSCs require clarification. Also unclear is how increase of alveolar ATP repairs the alveolar epithelial and endothelial barriers that are pathologically defective in ALI. Although these issues require attention, we propose that exogenous BMSCs provide a "Trojan horse" therapeutic strategy for supplying fresh mitochondria to injured cells, thereby enhancing cellular bioenergetics and improving organ function in ALI and other inflammatory diseases.