James Devaney

Background: Ventilator-induced lung injury (VILI) frequently worsens acute respiratory distress syndrome (ARDS) severity. Human mesenchymal stem/stromal cells (MSCs) offer considerable therapeutic promise, but the key impediments of clinical translation stem from limitations due to cell source and availability, and concerns regarding the loss of efficacy following cryopreservation. These experiments compared the efficacy of umbilical-cord-derived MSCs (UC-MSCs), a readily available and homogenous tissue source, to the previously more widely utilised bone-marrow-derived MSCs (BM-MSCs). We assessed their capacity to limit inflammation, resolve injury and enhance repair in relevant lung mechanical stretch models, and the impact of cryopreservation on therapeutic efficacy. Methods: In series 1, confluent alveolar epithelial layers were subjected to cyclic mechanical stretch (22% equibiaxial strain) and wound injury, and the potential of the secretome from BM- and UC-derived MSCs to attenuate epithelial inflammation and cell death, and enhance wound repair was determined. In series 2, anesthetized rats underwent VILI, and later received, in a randomised manner, 1 × 10⁷ MSCs/kg intravenously, that were: (i) fresh BM-MSCs, (ii) fresh UC-MSCs or (iii) cryopreserved UC-MSCs. Control animals received a vehicle (PBS). The extent of the resolution of inflammation and injury, and repair was measured at 24 h. Results: Conditioned medium from BM-MSCs and UC-MSCs comparably decreased stretch-induced pulmonary epithelial inflammation and cell death. BM-MSCs and UC-MSCs comparably enhanced wound resolution. In animals subjected to VILI, both fresh BM-MSCs and UC-MSCs enhanced injury resolution and repair, while cryopreserved UC-MSCs comparably retained their efficacy. Conclusions: Cryopreserved UC-MSCs can reduce stretch-induced inflammation and cell death, enhance wound resolution, and enhance injury resolution and repair following VILI. Cryopreserved UC-MSCs represent a more abundant, cost-efficient, less variable and equally efficacious source of therapeutic MSC product.

Objective: Although mesenchymal stem/stromal cells represent a promising therapeutic strategy for acute respiratory distress syndrome, clinical translation faces challenges, including scarcity of bone marrow donors, and reliance on bovine serum during mesenchymal stem/stromal cell proliferation. We wished to compare mesenchymal stem/stromal cells from human umbilical cord, grown in xeno-free conditions, with mesenchymal stem/stromal cells from human bone marrow, in a rat model of Escherichia coli pneumonia. In addition, we wished to determine the potential for umbilical cord-mesenchymal stem/stromal cells to reduce E. coli–induced oxidant injury. Design: Randomized animal study. Setting: University research laboratory. Subjects: Male Sprague-Dawley rats. Interventions: Acute respiratory distress syndrome was induced in rats by intratracheal instillation of E. coli (1.5–2 × 10⁹ CFU/kg). “Series 1” compared the effects of freshly thawed cryopreserved umbilical cord-mesenchymal stem/stromal cells with bone marrow-mesenchymal stem/stromal cells on physiologic indices of lung injury, cellular infiltration, and E. coli colony counts in bronchoalveolar lavage. “Series 2” examined the effects of cryopreserved umbilical cord-mesenchymal stem/stromal cells on survival, as well as measures of injury, inflammation and oxidant stress, including production of reactive oxidative species, reactive oxidative species scavenging by superoxide dismutase-1 and superoxide dismutase-2. Measurements and Main Results: In “Series 1,” animals subjected to E. coli pneumonia who received umbilical cord-mesenchymal stem/stromal cells had improvements in oxygenation, respiratory static compliance, and wet-to-dry ratios comparable to bone marrow-mesenchymal stem/stromal cell treatment. E. coli colony-forming units in bronchoalveolar lavage were reduced in both cell therapy groups, despite a reduction in bronchoalveolar lavage neutrophils. In series 2, umbilical cord-mesenchymal stem/stromal cells enhanced animal survival and decreased alveolar protein and proinflammatory cytokine concentrations, whereas increasing interleukin-10 concentrations. Umbilical cord-mesenchymal stem/stromal cell therapy decreased nicotinamide adenine dinucleotide phosphate-oxidase 2 and inducible nitric oxide synthase and enhanced lung concentrations of superoxide dismutase-2, thereby reducing lung tissue reactive oxidative species concentrations. Conclusions: Our results demonstrate that freshly thawed cryopreserved xeno-free human umbilical cord-mesenchymal stem/stromal cells reduce the severity of rodent E. coli–induced acute respiratory distress syndrome. Umbilical cord-mesenchymal stem/stromal cells, therefore, represent an attractive option for future clinical trials in acute respiratory distress syndrome.

The potential for mesenchymal stem cells (MSCs) to reduce the severity of experimental lung injury has been established in several pre-clinical studies. We have recently demonstrated that MSCs, and MSC-secreted factors (secretome), enhance lung repair and regeneration at 48 h following ventilation-induced lung injury (VILI). We wished to determine the potential for MSC therapy to exert beneficial effects in the early recovery phase following VILI when ongoing injury coexists with processes of repair, and to compare the efficacy of MSC therapy to the use of the secretome alone. Male Sprague–Dawley rats were anesthetized, oro-tracheally intubated, and subjected to high stretch mechanical ventilation until lung compliance had declined by 50 % of baseline. Animals were then weaned from mechanical ventilation, and anesthesia discontinued. Once awake and spontaneously ventilating, animals received an intravenous injection of either rodent MSCs (10 million/kg), MSC-conditioned medium, fibroblasts (10 million/kg), or vehicle. Thereafter, the animals were allowed to recover and the extent of lung injury/repair was determined after 4 h. Treatment with MSCs diminished injury and enhanced recovery following VILI to a greater extent compared to MSC-conditioned medium, with fibroblasts proving ineffective. MSCs, but not their conditioned medium, attenuated indices of lung injury including oxygenation, respiratory compliance, and lung edema. Total lung water as assessed by wet:dry ratio, bronchoalveolar lavage total inflammatory cell, neutrophil counts, and alveolar IL-6 concentrations were reduced in the animals that received MSC therapy. The immunomodulating and/or reparative effect of MSCs is evident early after VILI in this model. MSC-conditioned medium was not as effective as the cells themselves in diminishing injury and restoring lung structure and function.

Mesenchymal stromal cells (MSCs) demonstrate considerable promise in preclinical acute respiratory distress syndrome models. We wished to determine the efficacy and mechanisms of action of human MSCs (hMSCs) in the setting of acute lung injury induced by prolonged Escherichia coli pneumonia in the rat. Adult male Sprague Dawley rats underwent intratracheal instillation of E. coli bacteria in all experiments. In Series 1, animals were randomised to intravenous administration of: (1) vehicle (phosphate buffered saline (PBS), 300 μL); (2) 1×10(7) fibroblasts/kg; (3) 1×10(7) hMSCs/kg or (4) 2×10(7) hMSCs/kg. Series 2 determined the lowest effective hMSC dose. Series 3 compared the efficacy of intratracheal versus intravenous hMSC administration, while Series 4 examined the efficacy of cryopreserved hMSC. Series 5 examined the efficacy of the hMSC secretome. Parallel in vitro experiments further assessed the potential for hMSCs to secrete LL-37 and modulate macrophage phagocytosis. hMSC therapy reduced the severity of rodent E. coli pneumonia, improving survival, decreasing lung injury, reducing lung bacterial load and suppressing inflammation. Doses as low as 5×10(6) hMSCs/kg were effective. Intratracheal hMSC therapy was as effective as intravenous hMSC. Cryopreserved hMSCs were also effective, while the hMSC secretome was less effective in this model. hMSC therapy enhanced macrophage phagocytic capacity and increased lung and systemic concentrations of the antimicrobial peptide LL37. hMSC therapy decreased E. coli induced pneumonia injury and reduced lung bacterial burden, potentially via enhanced macrophage phagocytosis and increased alveolar LL-37 concentrations.

Activation of the nuclear factor-κB (NF-κB) pathway is central to the pathogenesis of lung injury and inflammation. We determined whether targeted overexpression of inhibitor-κBα (IκBα) in the lung could decrease the severity of ventilator-induced lung injury (VILI). Anaesthetized adult male Sprague-Dawley rats were randomly allocated to undergo intratracheal instillation of: (i) vehicle alone (surfactant, n=10); (ii) 1×10(10) adeno-associated virus encoding IκBα (AAV-IκBα, n=10); (iii) 5×10(10) AAV-IκBα (n=10); and (iv) 1×10(10) AAV-Null (n=5). This was followed by 4 h of injurious mechanical ventilation. Subsequent experiments examined the effect of IκBα overexpression in animals undergoing 'protective' mechanical ventilation. IκBα overexpression increased survival duration at both the lower [3.8 h (0.4)] and higher [3.6 h (0.7)] doses compared with vehicle [2.7 h (1.0)] or the null transgene [2.2 h (0.8)]. IκBα overexpression reduced the alveolar-arterial oxygen gradient (kPa) at both the lower [53 (21)] and higher [52 (19)] doses compared with vehicle [75 (8.5)] or the null transgene [70 (15)], decreased alveolar neutrophil infiltration, and reduced alveolar concentrations of interleukin (IL)-1β and IL-10. The lower IκBα dose was as effective as the higher dose. IκBα overexpression had no effect in the setting of protective lung ventilation. Inhibition of pulmonary NF-κB activity by IκBα overexpression reduced the severity of VILI in a rat model.

Introduction: Nuclear factor (NF)-κB is central to the pathogenesis of inflammation in acute lung injury, but also to inflammation resolution and repair. We wished to determine whether overexpression of the NF-κB inhibitor IκBα could modulate the severity of acute and prolonged pneumonia-induced lung injury in a series of prospective randomized animal studies. Methods: Adult male Sprague-Dawley rats were randomized to undergo intratracheal instillation of (a) 5 × 10⁹ adenoassociated virus (AAV) vectors encoding the IκBα transgene (5 × 10⁹ AAV-IκBα); (b) 1 × 10¹⁰ AAV-IκBα; (c) 5 × 10¹⁰ AAV-IκBα; or (d) vehicle alone. After intratracheal inoculation with Escherichia coli, the severity of the lung injury was measured in one series over a 4-hour period (acute pneumonia), and in a second series after 72 hours (prolonged pneumonia). Additional experiments examined the effects of IκBα and null-gene overexpression on E. coli-induced and sham pneumonia. Results: In acute pneumonia, IκBα dose-dependently decreased lung injury, improving arterial oxygenation and lung static compliance, reducing alveolar protein leak and histologic injury, and decreasing alveolar IL-1β concentrations. Benefit was maximal at the intermediate (1 × 10¹⁰) IκBα vector dose; however, efficacy was diminished at the higher (5 × 10¹⁰) IκBα vector dose. In contrast, IκBα worsened prolonged pneumonia-induced lung injury, increased lung bacterial load, decreased lung compliance, and delayed resolution of the acute inflammatory response. Conclusions: Inhibition of pulmonary NF-κB activity reduces early pneumonia-induced injury, but worsens injury and bacterial load during prolonged pneumonia.

Background: Mesenchymal stromal cells (MSCs) have been demonstrated to attenuate acute lung injury when delivered by intravenous or intratracheal routes. The authors aimed to determine the efficacy of and mechanism of action of intratracheal MSC therapy and to compare their efficacy in enhancing lung repair after ventilation-induced lung injury with intravenous MSC therapy. Methods: After induction of anesthesia, rats were orotracheally intubated and subjected to ventilation-induced lung injury (respiratory rate 18 min⁻¹, P _insp 35 cm H₂O,) to produce severe lung injury. After recovery, animals were randomized to receive: (1) no therapy, n = 4; (2) intratracheal vehicle (phosphate-buffered saline, 300 µl, n = 8); (3) intratracheal fibroblasts (4 × 10⁶ cells, n = 8); (4) intratracheal MSCs (4 × 10⁶ cells, n = 8); (5) intratracheal conditioned medium (300 µl, n = 8); or (6) intravenous MSCs (4 × 10⁶ cells, n = 4). The extent of recovery after acute lung injury and the inflammatory response was assessed after 48 h. Results: Intratracheal MSC therapy enhanced repair after ventilation-induced lung injury, improving arterial oxygenation (mean ± SD, 146 ± 3.9 vs. 110.8 ± 21.5 mmHg), restoring lung compliance (1.04 ± 0.11 vs. 0.83 ± 0.06 ml·cm H₂O⁻¹), reducing total lung water, and decreasing lung inflammation and histologic injury compared with control. Intratracheal MSC therapy attenuated alveolar tumor necrosis factor-α (130 ± 43 vs. 488 ± 211 pg·ml⁻¹) and interleukin-6 concentrations (138 ± 18 vs. 260 ± 82 pg·ml⁻¹). The efficacy of intratracheal MSCs was comparable with intravenous MSC therapy. Intratracheal MSCs seemed to act via a paracine mechanism, with conditioned MSC medium also enhancing lung repair after injury. Conclusions: Intratracheal MSC therapy enhanced recovery after ventilation-induced lung injury via a paracrine mechanism, and was as effective as intravenous MSC therapy.

Purpose Superoxide is produced by activated neutrophils during the inflammatory response to stimuli such as endotoxin, can directly or indirectly injure host cells, and has been implicated in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We wished to determine the potential for pulmonary overexpression of the extracellular isoform of superoxide dismutase (EC-SOD) to reduce the severity of endotoxin-induced lung injury. Methods Animals were randomly allocated to undergo intratracheal instillation of (1) surfactant alone (vehicle); (2) adeno-associated virus (AAV) vectors containing a null transgene (AAV-null); and (3) adeno-associated virus vectors containing the EC-SOD transgene (AAV-EC-SOD) and endotoxin was subsequently administered intratracheally. Two additional groups were randomized to receive (1) vehicle or (2) AAV-EC-SOD, and to undergo sham (vehicle) injury. The severity of the lung injury was assessed in all animals 24 h later. Results Endotoxin produced a severe lung injury compared to sham injury. The AAV vector encoding EC-SOD increased lung EC-SOD concentrations, and enhanced the antioxidant capacity of the lung. EC-SOD overexpression decreased the severity of endotoxin-induced ALI, reducing the decrement in systemic oxygenation and lung compliance, decreasing lung permeability and decreasing histologic injury. EC-SOD attenuated pulmonary inflammation, decreased bronchoalveolar lavage neutrophil counts, and reduced interleukin-6 and CINC-1 concentrations. The AAV vector itself did not contribute to inflammation or to lung injury. Conclusions Pulmonary overexpression of EC-SOD protects the lung against endotoxin-induced ALI.