Rudolf K. Braun

Effects of Chronic Intermittent Hypoxia on Allergen-Induced Airway Inflammation in Rats

Obstructive sleep apnea aggravates asthma, but its mechanisms are unknown. Chronic intermittent hypoxia is one hallmark feature of sleep apnea. In this study, we tested the effects of chronic intermittent hypoxia on allergen-induced inflammation in rats. Four groups (n = 9-11/group) of ovalbumin (OVA)-sensitized Brown-Norway rats underwent intermittent hypoxia (10% oxygen, 30 cycles/h, 10 h/d) or normoxia for 30 days concurrent with weekly OVA or vehicle challenges. Lung physiology, differential leukocyte counts from bronchoalveolar lavage, and histology (Picro Sirius Red staining for collagen content) were compared between groups 2 days after the last challenge. Gene expression in bronchoalveolar lavage cells was quantified by quantitative PCR. Compared with normoxia, chronic intermittent hypoxia reduced the FEV0.1/FVC ratio (P = 0.005), peak expiratory flow (P = 0.002), and mean midexpiratory flow (P = 0.004), predominantly in medium and large airways; decreased the baseline eosinophil number (P = 0.01) and amplified the effect of OVA on monocyte number (P = 0.02 for the interaction); in proximal airways, increased (P = 0.008), whereas in distal airways it decreased (P = 0.004), collagen density; induced qualitative emphysematous changes in lung periphery; and increased expression of the M2 macrophage marker YM-1 and augmented OVA-induced expression of plasminogen activator inhibitor-1. Chronic intermittent hypoxia alters immune response to allergen toward a more TH-1-predominant cellular phenotype with collagen deposition and matrix degradation, leading to airflow limitation. These findings highlight the potential of sleep apnea to aggravate airway dysfunction in patients with preexistent asthma.

Postnatal Hyperoxia Exposure Durably Impairs Right Ventricular Function and Mitochondrial Biogenesis

&NA; Prematurity complicates 12% of births, and young adults with a history of prematurity are at risk to develop right ventricular (RV) hypertrophy and impairment. The long‐term risk for pulmonary vascular disease, as well as mechanisms of RV dysfunction and ventricular‐vascular uncoupling after prematurity, remain poorly defined. Using an established model of prematurity‐related lung disease, pups from timed‐pregnant Sprague Dawley rats were randomized to normoxia or hyperoxia (fraction of inspired oxygen, 0.85) exposure for the first 14 days of life. After aging to 1 year in standard conditions, rats underwent hemodynamic assessment followed by tissue harvest for biochemical and histological evaluation. Aged hyperoxia‐exposed rats developed significantly greater RV hypertrophy, associated with a 40% increase in RV systolic pressures. Although cardiac index was similar, hyperoxia‐exposed rats demonstrated a reduced RV ejection fraction and significant RV‐pulmonary vascular uncoupling. Hyperoxia‐exposed RV cardiomyocytes demonstrated evidence of mitochondrial dysregulation and mitochondrial DNA damage, suggesting potential mitochondrial dysfunction as a cause of RV dysfunction. Aged rats exposed to postnatal hyperoxia recapitulate many features of young adults born prematurely, including increased RV hypertrophy and decreased RV ejection fraction. Our data suggest that postnatal hyperoxia exposure results in mitochondrial dysregulation that persists into adulthood with eventual RV dysfunction. Further evaluation of long‐term mitochondrial function is warranted in both animal models of premature lung disease and in human adults who were born preterm.

Biodistribution and Clearance of Human Mesenchymal Stem Cells by Quantitative Three-Dimensional Cryo-Imaging After Intravenous Infusion in a Rat Lung Injury Model

Cell tracking is a critical component of the safety and efficacy evaluation of therapeutic cell products. To date, cell‐tracking modalities have been hampered by poor resolution, low sensitivity, and inability to track cells beyond the shortterm. Three‐dimensional (3D) cryo‐imaging coregisters fluorescent and bright‐field microcopy images and allows for single‐cell quantification within a 3D organ volume. We hypothesized that 3D cryo‐imaging could be used to measure cell biodistribution and clearance after intravenous infusion in a rat lung injury model compared with normal rats. A bleomycin lung injury model was established in Sprague‐Dawley rats (n = 12). Human mesenchymal stem cells (hMSCs) labeled with QTracker655 were infused via jugular vein. After 2, 4, or 8 days, a second dose of hMSCs labeled with QTracker605 was infused, and animals were euthanized after 60, 120, or 240 minutes. Lungs, liver, spleen, heart, kidney, testis, and intestine were cryopreserved, followed by 3D cryo‐imaging of each organ. At 60 minutes, 82% ± 9.7% of cells were detected; detection decreased to 60% ± 17% and 66% ± 22% at 120 and 240 minutes, respectively. At day 2, 0.06% of cells were detected, and this level remained constant at days 4 and 8 postinfusion. At 60, 120, and 240 minutes, 99.7% of detected cells were found in the liver, lungs, and spleen, with cells primarily retained in the liver. This is the first study using 3D cryo‐imaging to track hMSCs in a rat lung injury model. hMSCs were retained primarily in the liver, with fewer detected in lungs and spleen.

Cardiopulmonary and histological characterization of an acute rat lung injury model demonstrating safety of mesenchymal stromal cell infusion

BACKGROUND AIMS In the field of cellular therapy, potential cell entrapment in the lungs following intravenous administration in a compromised or injured pulmonary system is an important concern that requires further investigation. We developed a rat model of inflammatory and fibrotic lung disease to mimic the human clinical condition of obliterative bronchiolitis (OB) and evaluate the safety of intravenous infusion of mesenchymal stromal cells (MSCs). This model was used to obtain appropriate safety information and functional characterization to support the translation of an ex vivo-generated cellular product into human clinical trials. To overcome spontaneous recovery and size limitations associated with current animal models, we used a novel multiple dose bleomycin strategy to induce lasting lung injury in rats. METHODS Intratracheal instillation of bleomycin was administered to rats on multiple days. MSCs were intravenously infused 7 days apart. Detailed pulmonary function tests including forced expiratory volume, total lung capacity, and invasive hemodynamic measurements were conducted to define the representative disease model and monitor cardiopulmonary hemodynamic consequences of the cell infusion. Post-euthanasia assessments included a thorough evaluation of lung morphology and histopathology. RESULTS The double dose bleomycin instillation regimen resulted in severe and irreversible lung injury and fibrosis. Cardiopulmonary physiological monitoring reveled that no adverse events could be attributed to the cell infusion process. DISCUSSION Although our study did not show the infusion of MSCs to result in an improvement in lung function or rescue of damaged tissue this study does confirm the safety of MSC infusion into damaged lungs.

Chronic intermittent hypoxia worsens bleomycin-induced lung fibrosis in rats

Obstructive sleep apnea (OSA) has been linked to increased mortality in pulmonary fibrosis. Its key feature, chronic intermittent hypoxia (CIH), can lead to oxidative stress and inflammation, known to lead to fibrotic pathology in other organs. We tested the effects of CIH in an animal model of bleomycin-induced lung fibrosis. Sprague Dawley rats were instilled intratracheally with bleomycin (Blm) or saline (Sal), and exposed to CIH or normal air (Norm) for 9 or 30 days. Pulmonary function was tested and lungs were harvested for histological and molecular analyses. In Blm-treated animals, 30days of CIH compared to Norm increased total lung collagen content (p=0.008) and reduced Quasi-static lung compliance (p=0.04). CIH upregulated lipid peroxidation and increased NF-κB activation, IL-17 mRNA and Col1α1 mRNA expression. Our results indicate that following Blm-induced lung injury, CIH amplifies collagen deposition via oxidative and inflammatory pathways, culminating in stiffer lungs. Thus, OSA may augment fibrosis in patients with interstitial lung disease.

Sex-Specific Skeletal Muscle Fatigability and Decreased Mitochondrial Oxidative Capacity in Adult Rats Exposed to Postnatal Hyperoxia

Premature birth affects more than 10% of live births, and is characterized by relative hyperoxia exposure in an immature host. Long-term consequences of preterm birth include decreased aerobic capacity, decreased muscular strength and endurance, and increased prevalence of metabolic diseases such as type 2 diabetes mellitus. Postnatal hyperoxia exposure in rodents is a well-established model of chronic lung disease of prematurity, and also recapitulates the pulmonary vascular, cardiovascular, and renal phenotype of premature birth. The objective of this study was to evaluate whether postnatal hyperoxia exposure in rats could recapitulate the skeletal and metabolic phenotype of premature birth, and to characterize the subcellular metabolic changes associated with postnatal hyperoxia exposure, with a secondary aim to evaluate sex differences in this model. Compared to control rats, male rats exposed to 14 days of postnatal hyperoxia then aged to 1 year demonstrated higher skeletal muscle fatigability, lower muscle mitochondrial oxidative capacity, more mitochondrial damage, and higher glycolytic enzyme expression. These differences were not present in female rats with the same postnatal hyperoxia exposure. This study demonstrates detrimental mitochondrial and muscular outcomes in the adult male rat exposed to postnatal hyperoxia. Given that young adults born premature also demonstrate skeletal muscle dysfunction, future studies are merited to determine whether this dysfunction as well as reduced aerobic capacity is due to reduced mitochondrial oxidative capacity and metabolic dysfunction.

Altered Right Ventricular Mechanical Properties Are Afterload Dependent in a Rodent Model of Bronchopulmonary Dysplasia

Infants born premature are at increased risk for development of bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), and ultimately right ventricular (RV) dysfunction, which together carry a high risk of neonatal mortality. However, the role alveolar simplification and abnormal pulmonary microvascular development in BPD affects RV contractile properties is unknown. We used a rat model of BPD to examine the effect of hyperoxia-induced PH on RV contractile properties. We measured in vivo RV pressure as well as passive force, maximum Ca2+ activated force, calcium sensitivity of force (pCa50) and rate of force redevelopment (ktr) in RV skinned trabeculae isolated from hearts of 21-and 35-day old rats pre-exposed to 21% oxygen (normoxia) or 85% oxygen (hyperoxia) for 14 days after birth. Systolic and diastolic RV pressure were significantly higher at day 21 in hyperoxia exposed rats compared to normoxia control rats, but normalized by 35 days of age. Passive force, maximum Ca2+ activated force, and calcium sensitivity of force were elevated and cross-bridge cycling kinetics depressed in 21-day old hyperoxic trabeculae, whereas no differences between normoxic and hyperoxic trabeculae were seen at 35 days. Myofibrillar protein analysis revealed that 21-day old hyperoxic trabeculae had increased levels of beta-myosin heavy chain (β-MHC), atrial myosin light chain 1 (aMLC1; often referred to as essential light chain), and slow skeletal troponin I (ssTnI) compared to age matched normoxic trabeculae. On the other hand, 35-day old normoxic and hyperoxic trabeculae expressed similar level of α- and β-MHC, ventricular MLC1 and predominantly cTnI. These results suggest that neonatal exposure to hyperoxia increases RV afterload and affect both the steady state and dynamic contractile properties of the RV, likely as a result of hyperoxia-induced expression of β-MHC, delayed transition of slow skeletal TnI to cardiac TnI, and expression of atrial MLC1. These hyperoxia-induced changes in contractile properties are reversible and accompany the resolution of PH with further developmental age, underscoring the importance of reducing RV afterload to allow for normalization of RV function in both animal models and humans with BPD.

Passenger Leukocytes Revisited: One Passenger That Refuses to Leave the Airspace

Passenger leukocytes, the leukocyte compartment contained within tissue and organ transplants, have long been known to have both immunogenic (1,2) and tolerogenic (3,4) properties. The interesting report by Nayak et al (5) asserts that while there are many different types of passenger cells in the bronchoalveolar lavage (BAL) fluid from a transplanted lung, the alveolar macrophage (AMAC) lineage, represented by autofluorescent cells expressing CD45, CD11b, HLA-DR, the lectin CD169 (Siglec-1), the mannose receptor CD206, and the scavenger receptor CD163, does not undergo replacement by recipient cells but rather persists wholly as a donor leukocyte compartment within the airspaces of the transplanted organ. Previously, these persisting AMACs have been marginalized or ignored because in the transplant setting, they constitute only about 7% of the total BAL cells typically available via routine saline (PBS) lavage. By using EDTA to dislodge firmly attached cells, the authors have found that the vast majority of the BAL cells in the normal lung in mice belong to this self-renewing population. Clinical practice would not permit EDTA use in BAL performed in human subjects, so endogenous AMACs are undoubtedly poorly represented when BAL is performed in the posttransplant human lung. However, by the use of this extensive panel of markers to identify the true AMAC population, it will be possible going forward to examine not only the retention of these cells but also their functional capacity. Another aspect of great interest in the report by Nayak et al (5) is the use of monoclonal antibodies specific for HLA-A2(BB7.2) and HLA-A3 (GAP.A3) to definitively localize donor vs recipient cells to the AMAC lineage. Using this approach, the authors find that rather than being turned over and replaced by recipient bone marrow–derived cells, AMACs persist primarily as donor type cells up to 3.5 years posttransplantion, with little evidence that recipient AMACs have infiltrated into this donor AMAC population.

The Role of Autoimmunity in the Pathogenesis of Obliterative Bronchiolitis

Many risk factors and post-transplant events have been linked to the development of bronchiolitis obliterans syndrome. Evolving research suggests that the development of cell-mediated and humoral reactivity to self-antigens (collagen V, K-α1 tubulin) in the lung allograft may play a very significant role in the bronchiolar inflammation and fibrosis that lead to obliterative bronchiolitis and progressive graft dysfunction and loss. Alloimmune and autoimmune mechanisms likely work together to mediate chronic lung allograft rejection. This chapter examines the role of autoimmunity in bronchiolitis obliterans syndrome with a focus on the role of Th17 lymphocytes, IL-17, and immune regulatory mechanisms in the development and progression of obliterative bronchiolitis.