Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven 'postnatal vasculogenesis' has been suggested as an ...important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1-2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2-3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell-specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human lung diseases, providing newer insights into their pathophysiological mechanisms.
Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene ...therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.
Mortality and morbidity of acute lung injury and acute respiratory distress syndrome remain high because of the lack of pharmacological therapies to prevent injury or promote repair. Mesenchymal stem ...cells (MSCs) prevent lung injury in various experimental models, despite a low proportion of donor-derived cell engraftment, suggesting that MSCs exert their beneficial effects via paracrine mechanisms. We hypothesized that soluble factors secreted by MSCs promote the resolution of lung injury in part by modulating alveolar macrophage (AM) function. We tested the therapeutic effect of MSC-derived conditioned medium (CdM) compared with whole MSCs, lung fibroblasts, and fibroblast-CdM. Intratracheal MSCs and MSC-CdM significantly attenuated lipopolysaccharide (LPS)-induced lung neutrophil influx, lung edema, and lung injury as assessed by an established lung injury score. MSC-CdM increased arginase-1 activity and Ym1 expression in LPS-exposed AMs. In vivo, AMs from LPS-MSC and LPS-MSC CdM lungs had enhanced expression of Ym1 and decreased expression of inducible nitric oxide synthase compared with untreated LPS mice. This suggests that MSC-CdM promotes alternative macrophage activation to an M2 "healer" phenotype. Comparative multiplex analysis of MSC- and fibroblast-CdM demonstrated that MSC-CdM contained several factors that may confer therapeutic benefit, including insulin-like growth factor I (IGF-I). Recombinant IGF-I partially reproduced the lung protective effect of MSC-CdM. In summary, MSCs act through a paracrine activity. MSC-CdM promotes the resolution of LPS-induced lung injury by attenuating lung inflammation and promoting a wound healing/anti-inflammatory M2 macrophage phenotype in part via IGF-I.
Background Bronchopulmonary dysplasia (BPD) remains a main complication of extreme prematurity and currently lacks efficient treatment. Rat bone marrow-derived mesenchymal stem cells (MSC) prevent ...lung injury in an oxygen-induced model of BPD. Human cord is an advantageous source of stem cells that is especially appealing for the treatment of neonatal diseases. The therapeutic benefit after established lung injury and long-term safety of cord-derived stem cells is unknown. Methods Human cord-derived perivascular cells (PCs) or cord blood-derived MSCs were delivered prophylactically or after established alveolar injury into the airways of newborn rats exposed to hyperoxia, a well-established BPD model. Results Rat pups exposed to hyperoxia showed the characteristic arrest in alveolar growth with air space enlargement and loss of lung capillaries. PCs and MSCs partially prevented and rescued lung function and structure. Despite therapeutic benefit, cell engraftment was low, suggesting that PCs and MSCs act via a paracrine effect. Accordingly, cell free-derived conditioned media from PCs and MSCs also exerted therapeutic benefit when used either prophylactically or therapeutically. Finally, long-term (6 months) assessment of stem cell or conditioned media therapy showed no adverse lung effects of either strategy, with persistent improvement in exercise capacity and lung structure. Conclusions Human umbilical cord-derived PCs and MSCs exert short- and long-term therapeutic benefit without adverse lung effects in this experimental model and offer new therapeutic options for lung diseases characterised by alveolar damage.
BACKGROUND—Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. ...Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth.
METHODS AND RESULTS—Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth–arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood–derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension.
CONCLUSIONS—Impaired ECFC function may contribute to arrested alveolar growth. Cord blood–derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage.
Bronchopulmonary dysplasia (BPD), the most common complication of prematurity, arises from various factors that compromise lung development, including oxygen and inflammation. Hyperoxia has been used ...to mimic the disease in newborn rodents. The use of a second hit to induce systemic inflammation has been suggested as an added strategy to better mimic the inflammatory aspect of BPD. Here we report a novel 2 hit (2HIT) BPD model with in-depth characterization of the innate immune response, enabling mechanistic studies of therapies with an immunomodulatory component.
C57BL/6N mice were exposed to 85% O
from postnatal day (P)1 to P7, and received postnatally (P3) Escherichia coli LPS. At various timepoints, immune activation in the lung and at the systemic level was analyzed by fluorescence-activated cell sorting (FACS), and gene and protein expressions.
2HIT mice showed fewer alveoli, increased lung compliance, and right ventricular hypertrophy. A transient proinflammatory cytokine response was observed locally and systemically. Type 2 anti-inflammatory cytokine expression was decreased in the lung together with the number of mature alveolar macrophages. Simultaneously, a Siglec-F intermediate macrophage population emerged.
This study provides long-term analysis of the 2HIT model, suggesting impairment of type 2 cytokine environment and altered alveolar macrophage profile in the lung.
We have developed a novel 2HIT mouse BPD model with postnatal LPS and hyperoxia exposure, which enables mechanistic studies of potential therapeutic strategies with an immunomodulatory component. This is the first report of in-depth characterization of the lung injury and recovery describing the evolution of the innate immune response in a standardized mouse model for experimental BPD with postnatal LPS and hyperoxia exposure. The 2HIT model has the potential to help understand the link between inflammation and impaired lung development, and will enable testing of new therapies in a short and more robust manner.
Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung ...disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we show that hyperoxia-induced experimental BPD in newborn mice led to lifelong impairments in cerebrovascular structure and function as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing nonhuman primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Together, our findings establish a relationship between BPD and abnormal neurodevelopmental outcomes and identify molecular and cellular players of neonatal brain injury that persist throughout adulthood that may be targeted for early intervention to aid this vulnerable patient population.
Lung hypoplasia and persistent pulmonary hypertension of the newborn limit survival in congenital diaphragmatic hernia (CDH). Unlike other diseases resulting in persistent pulmonary hypertension of ...the newborn, infants with CDH are refractory to inhaled nitric oxide (NO). Nitric oxide mediates pulmonary vasodilatation at birth in part via cyclic GMP production. Phosphodiesterase type 5 (PDE5) limits the effects of NO by inactivation of cyclic GMP. Because of the limited success in postnatal management of CDH, we hypothesized that antenatal PDE5 inhibition would attenuate pulmonary artery remodeling in experimental nitrofen-induced CDH.
Nitrofen administered at embryonic day 9.5 to pregnant rats resulted in a 60% incidence of CDH in the offspring and recapitulated features seen in human CDH, including structural abnormalities (lung hypoplasia, decreased pulmonary vascular density, pulmonary artery remodeling, right ventricular hypertrophy), and functional abnormalities (decreased pulmonary artery relaxation in response to the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide). Antenatal sildenafil administered to the pregnant rat from embryonic day 11.5 to embryonic day 20.5 crossed the placenta, increased fetal lung cyclic GMP and decreased active PDE5 expression. Antenatal sildenafil improved lung structure, increased pulmonary vessel density, reduced right ventricular hypertrophy, and improved postnatal NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide-induced pulmonary artery relaxation. This was associated with increased lung endothelial NO synthase and vascular endothelial growth factor protein expression. Antenatal sildenafil had no adverse effect on retinal structure/function and brain development.
Antenatal sildenafil improves pathological features of persistent pulmonary hypertension of the newborn in experimental CDH and does not alter the development of other PDE5-expressing organs. Given the high mortality/morbidity of CDH, the potential benefit of prenatal PDE5 inhibition in improving the outcome for infants with CDH warrants further studies.
Sepsis is the main cause of morbidity and mortality in neonates. Mesenchymal stromal cells (MSCs) are potent immune-modulatory cells. Their effect in neonatal sepsis has never been explored. We ...hypothesized that human umbilical cord-derived MSCs (hUC-MSCs) improve survival in experimental neonatal sepsis. Sepsis was induced in 3-day-old rats by intravenous injection of Escherichia coli (5 × 10
/rat). One hour after infection, rats were treated intravenously with normal saline, hUC-MSCs, or with interferon-γ preconditioned hUC-MSCs (10
cells/kg). Eighteen hours after infection, survival, bacterial counts, lung neutrophil and macrophage influx, phagocytosis and apoptosis of splenocytes plasma, and LL-37 concentration were evaluated. Animals were observed for survival for 72 h after E. coli injection. Treatment with either hUC-MSCs or preconditioned hUC-MSCs significantly increased survival (hUC-MSCs, 81%; preconditioned hUC-MSCs, 89%; saline, 51%; P < 0.05). Both hUC-MSCs and preconditioned hUC-MSCs enhanced bacterial clearance. Lung neutrophil influx was decreased with preconditioned hUC-MSCs. The number of activated macrophages (CD206
) in the spleen was increased with hUC-MSCs and preconditioned hUC-MSCs; preconditioned hUC-MSCs increased the phagocytic activity of CD206
macrophages. hUC-MSCs and preconditioned hUC-MSCs decreased splenocyte apoptosis in E. coli infected rats. Finally, LL-37 plasma levels were elevated in neonatal rats treated with hUC-MSCs or preconditioned hUC-MSCs. hUC-MSCs enhance survival and bacterial clearance in experimental neonatal sepsis. hUC-MSCs may be an effective adjunct therapy to reduce neonatal sepsis-related morbidity and mortality.
Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary ...hypertension (PHT). Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S), carbon monoxide and nitric oxide (NO), belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD.
We took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate) to study its lung protective potential in vitro and in vivo.
In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation.
H2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.
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