Dr. Mc Cully and his colleagues reported the first clinical application of mitochondrial transplantation in pediatric patients with ischemia-reperfusion-associated myocardial dysfunction16. Mitochondria transplantation have since been considered as a revolutionary approach for regenerative medicine22. Mitochondrial transplantation has the advantage of rapid isolation and purification to meet the clinical needs23. Mitochondria are unlikely to induce alloreactivity and damage-associated molecular pattern molecules reaction. Therefore, mitochondria have potential for both syngeneic and allogeneic transplants24. Furthermore, exogenous mitochondria can be conveniently administrated through intravenous route14, 18.
Intratracheal lipopolysaccharide instillation destroys lung parenchyma through the generation of proteases and reactive oxygen and nitrogen species produced by activated polymorphonuclear cells in the interstitial and alveolar compartments that is comparable to the pathophysiological changes in patients with ALI/ARDS25. Previous studies found that the intra-alveolar inflammatory reaction composed of a neutrophilic exudate at the initial 6 to 12 h, a monocytic exudate peaking at 24 h, and then followed by a lymphocytic exudate26. After lipopolysaccharide instillation, we detected hypoxemia and hypercapnia, indicating the impaired gas exchange due to development of alveolar dead space and ventilation-to-perfusion mismatch in these animals27. The alveolar-capillary barrier is organized by a network of collagen and laminin that separates the epithelium and endothelium28. The capillary endothelium is a semipermeable barrier to fluid exchange, whereas the alveolar epithelium is a tight layer that restricts the passage of water, electrolytes and hydrophilic solutes to the air space29. Increased protein contents in the BALF, pulmonary hemorrhage, inflammatory cell infiltration, and parenchymal consolidation further confirmed alveolar-capillary disruption.
Under IVIS, we detected the engraftment of transplanted mitochondria into the pulmonary circulation was significantly more enhanced in lungs of rats with ALI. The uptake of these exogenous mitochondria was further confirmed by the increased ATP concentrations in the lungs. Consistent with our previous report18, these findings suggest that intravenous delivery of exogenous mitochondria can selectively locate and be taken up by areas of the pulmonary arterial system with mitochondrial dysfunction. After two mitochondrial transplants, the protein content recovered in BALF and extravasated Evans blue dye was significantly reduced, suggesting improved integrity of the monolayer capillary endothelium in preventing leakage of plasma proteins and albumin into the air-space19, 30. However, lung water content measured by LWDR was not differ significantly among the three treatment groups. We speculate that the development of extensive alveolar ectasia, lung consolidation and predominant infiltration of lymphocytes at 24 h after ALI31, might obscure the formation of pulmonary edema.
Pulmonary endothelial function was also assessed using vasomotor function tests and through the expression of eNOS in the pulmonary artery. The isometric tension analysis found no differences in the concentration response curves of phenylephrine and acetylcholine of ALI animals treated with PBS or mitochondria. However, the relaxation response to maximal concentration of acetylcholine, endothelial-dependent relaxation induced by stimulating the muscarinic receptors, was significantly potentiated in animals received mitochondrial treatment. The increased isometric tension in the pulmonary artery of ALI rats that received PBS at high acetylcholine concentrations might be due to vasoconstriction reaction mediated by direct stimulation of the muscarinic receptors on the vascular smooth muscle cells where the endothelial layer is damaged or denuded5, 32. Since the lung injury primarily originated from the bronchoalveolar site in this model, it was therefore reasonable that fewer changes in the vasoreactivity tests were detected in comparison to experimental models of direct pulmonary endothelial injury, such as intravenous administration of oleic acid5. Endothelial NOS is an important enzyme in the endothelial cells that synthesizes optimal amount of NO in order to maintain a normal endothelial homeostasis21, 33. Phosphorylation of eNOS at Ser1177 through Akt/Protein kinase B or AMP-activated protein kinase is a critical requirement for eNOS activation34. The Western blot analysis confirmed that mitochondria transplantation restored eNOS and p-eNOS-S1177 levels in the pulmonary artery, further supporting the engraftment of exogenous mitochondria into the pulmonary circulation can improve endothelial function in rats with ALI. In addition, the increase in capillary endothelium integrity also reduces trans-endothelial migration of immune cells towards the inflammatory cascade in lung tissues35. iNOS and CD11b are considered as the cell markers of activated M1 macrophages during the pro-inflammatory phase of ALI36. The significant suppression of iNOS and CD11b expressions in the injured lung following mitochondrial transplantation might imply the reduced pro-inflammatory cell infiltration in rats with ALI.
The exact mechanisms underlying mitochondrial transplantation in tissue regeneration remains undetermined, but three potential mechanisms have been proposed37. The Ca2+ buffering capacity of mitochondria may attenuate the environmental Ca2+ overload during cellular stress through the opening of voltage-dependent anion channels38. The second theory proposes that the internalization of exogenous mitochondria in recipient cells improves the mitochondrial function of target cells by increasing ATP generation and mitochondrial oxygen consumption39. Finally, the viable exogenous mitochondria may release ATP into the extracellular environment and salvage the dysfunctional cells37, 40. However, there is currently insufficient evidence to support the direct anti-inflammatory potential of transplanted mitochondria during tissue injury. It is then expected that we did not find any significant effects of mitochondrial transplant on the myeloperoxidase activity assay in the lung tissue exposed to endotoxin instillation.
There are a number of limitations in our study. First, the intracellular localization of these exogenous mitochondria was not determined in this study. Secondly, this study recorded very few ALI-related mortality throughout the study period. The effects of mitochondrial transplantation on other outcome measurements (e.g. overall mortality rate and lung parenchymal repair) were not studied. Thirdly, the optimal dose of mitochondria and the frequency of treatment were rather arbitrary, as there is still no general consensus on the standard dosing for mitochondrial transplantation41. Fourthly, this was a pre-clinical, proof-of-concept research for the potential application of mitochondrial transplantation in ALI/ARDS and more mechanistic analysis are still under investigation in our laboratory.
In conclusion, this is the first report demonstrating that intravenous transplantation of viable allogeneic mitochondria in rats with endotoxin-induced ALI significantly improves gas exchange by reducing alveolar-capillary barrier endothelial disruption. However, the effects on survival outcomes and long term recovery of pulmonary function in subjects with ALI after mitochondrial transplants require further investigation.