The mitochondrial calcium uniporter of pulmonary type 2 cells determines severity of acute lung injury.

Acute Lung Injury (ALI) due to inhaled pathogens causes high mortality. Underlying mechanisms are inadequately understood. Here, by optical imaging of live mouse lungs we show that a key mechanism is the viability of cytosolic Ca2+ buffering by the mitochondrial Ca2+ uniporter (MCU) in the lung's surfactant-secreting, alveolar type 2 cells (AT2). The buffering increased mitochondrial Ca2+ and induced surfactant secretion in wild-type mice, but not in mice with AT2-specific MCU knockout. In the knockout mice, ALI due to intranasal LPS instillation caused severe pulmonary edema and mortality, which were mitigated by surfactant replenishment prior to LPS instillation, indicating surfactant's protective effect against alveolar edema. In wild-type mice, intranasal LPS, or Pseudomonas aeruginosa decreased AT2 MCU. Loss of MCU abrogated buffering. The resulting mortality was reduced by spontaneous recovery of MCU expression, or by MCU replenishment. Enhancement of AT2 mitochondrial buffering, hence endogenous surfactant secretion, through MCU replenishment might be a therapy against ALI.

Molecular programs of fibrotic change in aging human lung.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging.

A three-dimensional model of human lung development and disease from pluripotent stem cells.

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

Efficient generation of lung and airway epithelial cells from human pluripotent stem cells.

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.

Cadherin selectivity filter regulates endothelial sieving properties.

The molecular basis of endothelial protein sieving, the critical vascular barrier function that restricts flow of large plasma proteins into tissues while allowing small molecules and water to pass, is not understood. Here, we address this issue using a novel assay to detect macromolecular penetrance at microdomains of endothelial adherens junctions. Adherens junctions, as detected by cadherin-GFP expression, were distributed in the cell perimeter as high- or low-density segments. Low but not high-density segments permitted penetrance of a 70-kDa fluorescent dextran, a molecule of equivalent size to albumin. Expression of a cadherin mutant that abrogates strand-swap adhesive binding in the cadherin EC1 ectodomain, or alternatively of an α-actinin-1 mutant that inhibits F-actin bundling, increased both cadherin mobility and 70 kDa dextran penetrance at high-density segments. These findings suggest that adhesive interactions in the cadherin EC1 domain, which underlie adherens junction structure, are critical determinants of endothelial macromolecular sieving.

Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury.

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

Activation of TNFR1 ectodomain shedding by mitochondrial Ca2+ determines the severity of inflammation in mouse lung microvessels.

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 Ca2+, and the purinergic receptor P2Y2 prolonged the response. Concomitantly, the proinflammatory receptor TNF-α receptor-1 (TNFR1) was shed from the endothelial surface. Inhibiting the mitochondrial Ca2+ 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 Ca2+-dependent increase of mitochondrial H2O2 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.

A new hypothesis for Ca2+ flows in skeletal muscle and its implications for other cell types.

We offer a new hypothesis to explain calcium flows in skeletal muscle cells. Our model accounts for the uptake of Ca2+ from the extracellular fluid, and the release of Ca2+ from the sarcoplasmic reticulum (SR/ER) (the endoplasmic reticulum in muscle is named sarcoplasmic reticulum); this has engendered difficulty in reviews encompassing both muscle and nonmuscle cells. Here we will typically refer to the organelle as ER, except when specifically discussing muscle cells. The broad consideration of two major, still unexplained properties of skeletal muscle function, namely excitation contraction coupling and capacitative calcium entry are accounted for in a unitary hypothesis. This model allows a reinterpretation of existing data, and points to areas where new investigation may be fruitful. While primarily aimed at explaining Ca2+ flows in skeletal muscle, we consider findings of other systems to explore the implications of this hypothesis for other cell types.

A mechanism for both capacitative Ca(2+) entry and excitation-contraction coupled Ca(2+) release by the sarcoplasmic reticulum of skeletal muscle cells.

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca(2+) uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca(2+) from cytosol to SR, and measurements of cytosolic Ca(2+). When Ca(2+) is added to Ca(2+)-free cells in the presence of Tg, the measured cytosolic Ca(2+) rises. This has been universally interpreted to mean that as SR Ca(2+) is depleted, exogenous Ca(2+) crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca(2+) in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca(2+) in the presence of Tg showed an increase in luminal Ca(2+) concentration in response to added exogenous Ca(2+). While we were able to reproduce the conventional demonstration of CCE-an increase of Ca(2+) in the cytosol in the presence of thapsigargin-we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca(2+) release channel, the ryanodine receptor (RyR). This was also unexpected if Ca(2+) enters the cytosol first. When Ca(2+) was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca(2+) and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca(2+) transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca(2+) inhibited its own uptake, a known feature of the RyR. We confirmed that Ca(2+) enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn(2+) in place of direct Ca(2+) measurements; we showed that Mn(2+) was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca(2+) flow in muscle cells. Our findings are entirely explained by a new model whereby Ca(2+) enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca(2+) subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca(2+) is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.