Xiaohui Fang

Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung

Recent studies have suggested that bone marrow-derived multipotent mesenchymal stem cells (MSCs) may have therapeutic applications in multiple clinical disorders including myocardial infarction, diabetes, sepsis, and hepatic and acute renal failure. Here, we tested the therapeutic capacity of human MSCs to restore alveolar epithelial fluid transport and lung fluid balance from acute lung injury (ALI) in an ex vivo perfused human lung preparation injured by E. coli endotoxin. Intra-bronchial instillation of endotoxin into the distal airspaces resulted in pulmonary edema with the loss of alveolar epithelial fluid transport measured as alveolar fluid clearance. Treatment with allogeneic human MSCs or its conditioned medium given 1 h following endotoxin-induced lung injury reduced extravascular lung water, improved lung endothelial barrier permeability and restored alveolar fluid clearance. Using siRNA knockdown of potential paracrine soluble factors, secretion of keratinocyte growth factor was essential for the beneficial effect of MSCs on alveolar epithelial fluid transport, in part by restoring amiloride-dependent sodium transport. In summary, treatment with allogeneic human MSCs or the conditioned medium restores normal fluid balance in an ex vivo perfused human lung injured by E. coli endotoxin.

Antibacterial Effect of Human Mesenchymal Stem Cells Is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram‐negative (Escherichia coli and Pseudomonas aeruginosa) and Gram‐positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram‐negative bacteria was the human cathelicidin antimicrobial peptide, hCAP‐18/LL‐37. Both m‐RNA and protein expression data showed that the expression of LL‐37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony‐forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL‐37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC‐treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)‐treated mice. Human bone marrow‐derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP‐18/ LL‐37. STEM CELLS 2010;28:2229–2238

Concise Review: Mesenchymal Stem Cells for Acute Lung Injury: Role of Paracrine Soluble Factors

Morbidity and mortality have declined only modestly in patients with clinical acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), despite extensive research into the pathophysiology. Current treatment remains primarily supportive with lung‐protective ventilation and a fluid conservative strategy. Pharmacologic therapies that reduce the severity of lung injury in preclinical models have not yet been translated to effective clinical treatment options. Consequently, further research in translational therapies is needed. Cell‐based therapy with mesenchymal stem cells (MSCs) is one attractive new therapeutic approach. MSCs have the capacity to secrete multiple paracrine factors that can regulate endothelial and epithelial permeability, decrease inflammation, enhance tissue repair, and inhibit bacterial growth. This review will focus on recent studies, which support the potential therapeutic use of MSCs in ALI/ARDS, with an emphasis on the role of paracrine soluble factors. STEM CELLS 2011;29:913–919

Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial

BACKGROUND No effective pharmacotherapy for acute respiratory distress syndrome (ARDS) exists, and mortality remains high. Preclinical studies support the efficacy of mesenchymal stem (stromal) cells (MSCs) in the treatment of lung injury. We aimed to test the safety of a single dose of allogeneic bone marrow-derived MSCs in patients with moderate-to-severe ARDS. METHODS The STem cells for ARDS Treatment (START) trial was a multicentre, open-label, dose-escalation, phase 1 clinical trial. Patients were enrolled in the intensive care units at University of California, San Francisco, CA, USA, Stanford University, Stanford, CA, USA, and Massachusetts General Hospital, Boston, MA, USA, between July 8, 2013, and Jan 13, 2014. Patients were included if they had moderate-to-severe ARDS as defined by the acute onset of the need for positive pressure ventilation by an endotracheal or tracheal tube, a PaO2:FiO2 less than 200 mm Hg with at least 8 cm H2O positive end-expiratory airway pressure (PEEP), and bilateral infiltrates consistent with pulmonary oedema on frontal chest radiograph. The first three patients were treated with low dose MSCs (1 million cells/kg predicted bodyweight [PBW]), the next three patients received intermediate dose MSCs (5 million cells/kg PBW), and the final three patients received high dose MSCs (10 million cells/kg PBW). Primary outcomes included the incidence of prespecified infusion-associated events and serious adverse events. The trial is registered with ClinicalTrials.gov, number NCT01775774. FINDINGS No prespecified infusion-associated events or treatment-related adverse events were reported in any of the nine patients. Serious adverse events were subsequently noted in three patients during the weeks after the infusion: one patient died on study day 9, one patient died on study day 31, and one patient was discovered to have multiple embolic infarcts of the spleen, kidneys, and brain that were age-indeterminate, but thought to have occurred before the MSC infusion based on MRI results. None of these severe adverse events were thought to be MSC-related. INTERPRETATION A single intravenous infusion of allogeneic, bone marrow-derived human MSCs was well tolerated in nine patients with moderate to severe ARDS. Based on this phase 1 experience, we have proceeded to phase 2 testing of MSCs for moderate to severe ARDS with a primary focus on safety and secondary outcomes including respiratory, systemic, and biological endpoints. FUNDING The National Heart, Lung, and Blood Institute.

Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1

Acute lung injury is characterized by injury to the lung epithelium that leads to impaired resolution of pulmonary edema and also facilitates accumulation of protein-rich edema fluid and inflammatory cells in the distal airspaces of the lung. Recent in vivo and in vitro studies suggest that mesenchymal stem cells (MSC) may have therapeutic value for the treatment of acute lung injury. Here we tested the ability of human allogeneic mesenchymal stem cells to restore epithelial permeability to protein across primary cultures of polarized human alveolar epithelial type II cells after an inflammatory insult. Alveolar epithelial type II cells were grown on a Transwell plate with an air-liquid interface and injured by cytomix, a combination of IL-1β, TNFα, and IFNγ. Protein permeability measured by 131I-labeled albumin flux was increased by 5-fold over 24 h after cytokine-induced injury. Co-culture of human MSC restored type II cell epithelial permeability to protein to control levels. Using siRNA knockdown of potential paracrine soluble factors, we found that angiopoietin-1 secretion was responsible for this beneficial effect in part by preventing actin stress fiber formation and claudin 18 disorganization through suppression of NFκB activity. This study provides novel evidence for a beneficial effect of MSC on alveolar epithelial permeability to protein.

Transforming Growth Factor-β1 Decreases Expression of the Epithelial Sodium Channel αENaC and Alveolar Epithelial Vectorial Sodium and Fluid Transport via an ERK1/2-dependent Mechanism

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-β1 (TGF-β1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-β1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-β1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-β1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-β1 also significantly decreased αENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the αENaC promoter in lung epithelial cells. The inhibitory effect of TGF-β1 on sodium uptake and αENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-β1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-β1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane αENaC expression mediated through an ERK1/2-dependent inhibition of the αENaC promoter activity.

Clinically relevant concentrations of β2-adrenergic agonists stimulate maximal cyclic adenosine monophosphate-dependent airspace fluid clearance and decrease pulmonary edema in experimental acid-induced lung injury*

Objective:To determine whether clinically relevant airspace concentrations of β2-adrenergic agonists stimulated maximal alveolar fluid clearance rates and to determine whether β2 agonist therapy decreased pulmonary edema in experimental acute lung injury. Design:Prospective randomized laboratory investigation. Setting:University-affiliated laboratory. Subjects:Sprague Dawley rats. Interventions:Dibutyryl cyclic adenosine monophosphate (cAMP), salmeterol, albuterol, and isoproterenol in normal rat lung. Salmeterol in a rat model of acid-induced lung injury. Measurements and Main Results:Basal alveolar fluid clearance was 7.6 ± 2.2 %/hr. Maximal cAMP-dependent alveolar fluid clearance rate was 32.9 ± 10.9 %/hr (p < .05). Racemic albuterol 10−5M, salmeterol 10−6M, and isoproterenol 10−6M each stimulated alveolar fluid clearance to a level comparable to maximal cAMP-dependent alveolar fluid clearance. Compared with basal rates, alveolar fluid clearance was increased by both racemic albuterol 10−6M (14.5 ± 3.0%, p < .05) and R-enantiomer 10−6M (15.0 ± 4.6%, p < .05), but there was no difference between the two groups. Intra-alveolar salmeterol 10−6M attenuated the degree of pulmonary edema following acid-induced lung injury. Extravascular lung water increased to only 180 ± 30 μL with salmeterol treatment, compared with 296 ± 65 μL in saline-treated rats 4 hrs after acid injury (p < .05). This decrease in lung water was accompanied by a 2.4-fold increase in the rate of alveolar fluid clearance at 4 hrs in the salmeterol-treated group. Lung endothelial permeability, expressed as extravascular plasma equivalents, was reduced to 64 ± 9 μL with salmeterol compared with 119 ± 51 μL in saline-treated rats 4 hrs after acid injury (p < .05). Conclusions:Clinically relevant airspace concentrations of β2-adrenergic agonists a) stimulate maximal cAMP-dependent airspace fluid clearance in normal lungs and b) reduce pulmonary edema in acid aspiration-induced lung injury by increasing alveolar fluid clearance and decreasing endothelial permeability. Clinical studies are required to determine whether β2-adrenergic agonists improve outcome in patients with acute lung injury.

Assessment of lungs rejected for transplantation and implications for donor selection

Present criteria for donor-lung selection exclude more than 85% of lungs. We aimed to establish if potentially suitable lungs are rejected for transplantation. We obtained 29 pairs of rejected lungs and assessed them by physiological, microbiological, and histological methods. Most donor lungs had no or mild pulmonary oedema (24/29 [83%]), intact alveolar fluid clearance (17/23 [74%]), and normal or mildly abnormal histological findings (18/29 [62%]). When all factors were considered, including microbiological and non-lung donor factors, 12 (41%) of 29 pairs of rejected lungs would have been potentially suitable for transplantation. Our findings emphasise the urgent need for prospective scientific assessment of selection of donors for lung transplantation.

Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis

Prior work has shown that transforming growth factor-β (TGF-β) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-β receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-β can mediate this process.

Evidence against aquaporin-1-dependent CO2 permeability in lung and kidney

AQP1‐dependent CO2 transport has been suggested from the increased CO2 permeability in Xenopus oocytes expressing AQP1. Potential implications of this finding include AQP1‐facilitated CO2 exchange in mammalian lung and HCO3−/CO2 transport in kidney proximal tubule. We reported previously that: (a) CO2 permeability in erythrocytes was not affected by AQP1 deletion, (b) CO2 permeability in liposomes was not affected by AQP1 reconstitution despite a 100‐fold increased water permeability, and (c) CO2 blow‐off by the lung in living mice was not impaired by AQP1 deletion. We extend these observations by direct measurement of CO2 permeabilities in lung and kidney. CO2 transport across the air‐space‐capillary barrier in isolated perfused lungs was measured from changes in air‐space fluid pH in response to addition/removal of HCO3−/CO2 from the pulmonary artery perfusate. The pH was measured by pleural surface fluorescence of a pH indicator (BCECF‐dextran) in the air‐space fluid. Air‐space fluid pH equilibrated rapidly (t1/2∼ 6 s) in response to addition/removal of HCO3−/CO2. However, the kinetics of pH change was not different in lungs of mice lacking AQP1, AQP5 or AQP1/AQP5 together, despite an up to 30‐fold reduction in water permeability. CO2 transport across BCECF‐loaded apical membrane vesicles from kidney proximal tubule was measured from the kinetics of intravesicular acidification in response to rapid mixing with a HCO3−/CO2 solution. Vesicles rapidly acidified (t1/2∼ 10 ms) in response to HCO3−/CO2 addition. However the acidification rate was not different in kidney vesicles from AQP1‐null mice despite a 20‐fold reduction in water permeability. The results provide direct evidence against physiologically significant transport of CO2 by AQP1 in mammalian lung and kidney.