Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration

Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. Whereas postnatal regeneration of alveolar tissue has been attributed to type II alveolar epithelial cells (AECII), we reasoned that bronchioalveolar stem cells (BASCs) have the potential to contribute substantially to this process. To test this hypothesis, unilateral pneumonectomy (PNX) was performed on adult female C57/BL6 mice to stimulate compensatory lung regrowth. The density of BASCs and AECII, and morphometric and physiological measurements, were recorded on days 1, 3, 7, 14, 28, and 45 after surgery. Vital capacity was restored by day 7 after PNX. BASC numbers increased by day 3, peaked to 220% of controls (P<0.05) by day 14, and then returned to baseline after active lung regrowth was complete, whereas AECII cell densities increased to 124% of baseline (N/S). Proliferation studies revealed significant BrdU uptake in BASCs and AECII within the first 7 days after PNX. Quantitative analysis using a systems biology model was used to evaluate the potential contribution of BASCs and AECII. The model demonstrated that BASC proliferation and differentiation contributes between 0 and 25% of compensatory alveolar epithelial (type I and II cell) regrowth, demonstrating that regeneration requires a substantial contribution from AECII. The observed cell kinetic profiles can be reconciled using a dual-compartment (BASC and AECII) proliferation model assuming a linear hierarchy of BASCs, AECII, and AECI cells to achieve lung regrowth.     

Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth

We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.     

Signals and mechanisms of compensatory lung growth.

Growth of the lung involves unique structure-function interactions not seen in solid organs. Mechanical feedback between the lung and thorax constitutes a major signal that sustains developmental as well as compensatory lung growth. After the loss of lung units as by pneumonectomy (PNX), increased mechanical stress and strain on the remaining units induce adaptive responses to augment oxygen transport, including 1) recruitment of alveolar-capillary reserves, 2) remodeling of existing tissue, and 3) regenerative growth of acinar tissue when strain exceeds a critical threshold. Alveolar hypoxia, hormones, and growth factors may feed into the mechanical feedback system to modify an existing growth response but are unlikely to initiate compensatory growth in the absence of sufficient mechanical signals. Whereas endogenous post-PNX alveolar growth preserves normal structure-function relationships, experimental manipulation of selected metabolic pathways can distort these relationships. Finally, PNX widens the disparity between the rapidly adapting acini and slowly adapting conducting airways and blood vessels, leading to disproportionate airflow and hemodynamic dysfunction and secondary hypertrophy of the right ventricle and respiratory muscles that limits overall organ function despite regeneration of gas exchange tissue. These are key concepts to consider when formulating approaches to stimulate or augment compensatory growth in chronic lung disease.     

Developmental signals do not further accentuate nonuniform postpneumonectomy compensatory lung growth.

Mechanical forces imposed on lung tissue constitute major stimuli for normal lung development and postpneumonectomy (PNX) compensatory growth and remodeling. Superimposing developmental signals on PNX signals augments compensatory alveolar growth but exaggerates airway-parenchymal dissociation (i.e., dysanaptic lung growth); the latter tends to offset benefits derived from the former. In adult dogs after PNX, lobar expansion and growth of the remaining lobes were markedly non-uniform (Ravikumar et al. J Appl Physiol 97:1567-1574, 2004). We hypothesized that superimposing developmental and post-PNX signals further accentuates nonuniformity of lobar growth. We used high-resolution computed tomography (HRCT) to follow regional lung expansion and growth in foxhounds undergoing right PNX at 2.5 mo of age compared with litter-matched control (Sham) animals; scans were performed 4 and 10 mo following surgery, i.e., before and after somatic maturity. Air and tissue volumes were measured in each lobe; tissue volume estimated by HRCT includes air-free tissue and blood in small vessels <1 mm. Interlobar nonuniformity of tissue volume was absent at 4 mo but evident 10 mo after PNX; growth of the remaining left lower lobe gradually lagged behind other lobes. At maturity, nonuniformity of lobar growth in pneumonectomized puppies was similar to that previously reported in pneumonectomized adults. We conclude that superimposing developmental and post-PNX signals enhances some aspects of compensatory lung growth and remodeling without altering its nonuniform spatial distribution.     

Regional lung growth following pneumonectomy assessed by computed tomography.

After pneumonectomy (PNX), mechanical strain on the remaining lung is greatly increased. To assess whether remaining lobes expand uniformly after left or right PNX (removing 42 and 58% of lung mass, respectively), we performed high-resolution computed tomography (CT) scans at 45 ml/kg above end-expiratory lung volume on adult male foxhounds after left or right PNX, which were compared with adult Sham controls. Air and tissue volumes were separately measured in each lobe. After left PNX, air and tissue volumes in the right upper and cardiac lobes increased approximately 2.2-fold above and below the heart, whereas volumes in right middle and lower lobes did not change significantly. After right PNX, air and tissue volumes in the left upper and middle lobes increased 2.3- to 2.7-fold across the midline anterior to the heart, whereas the left lower lobe expanded approximately 1.9-fold posterior to the heart. Regional changes in volume density of tissue post-PNX estimated by CT scan parallel postmortem estimates by morphometric analyses. Data indicate heterogeneous regional distribution of mechanical lung strain, which could influence the differential cellular compensatory response following right and left PNX.     

Variation in senescent-dependent lung changes in inbred mouse strains.

Previous studies from our laboratories showed lung development differences between inbred strains of mice. In the present study, the C57BL/6J (B6) and DBA/2J (D2) strains were examined for senescent-dependent differences with respect to the lung structure and function. Specifically, we hypothesize that senescent changes in lung vary between strains due to identifiable gene expression differences. Quasi-static pressure-volume curves and respiratory impedance measurements were performed on 2- and 20-mo-old B6 and D2 mice. Lung volume at 30 cm H(2)O (V(30)) pressure was significantly (P < 0.01) increased with age in both strains, but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. In addition, decreased elastic recoil pressure at 50% of V(30) and a reduction in airway resistance as a function of positive end-expiratory pressure were observed in 20-mo-old D2 mice but not in B6 mice. Morphometric analysis of lung parenchyma showed significant decreases in elastic fiber content with age in both strains, but the collagen content was significantly (P < 0.01) increased with age in D2 but not B6 mice at 20 mo. Furthermore, using quantitative RT-PCR methods, gene expression differences between strains suggested that D2 mice significantly (P < 0.05) downregulated the expressions of elastin (Eln) and procollagen I, III, and VI (Col1a1, Col3a1, and Col6a3) in lung tissue at 20 mo of age. These age-dependent changes were accompanied by an increased gene expression in matrix metalloproteinase 9 (Mmp9) in D2 and an increase in tissue inhibitor of matrix metalloproteinase (Timp1 and Timp4) in B6 mice. In conclusion, the results from the present study demonstrate that lung mechanics of both strains show significant age-dependent changes. However, changes in D2 mice are accelerated relative to B6 mice. Moreover, gene expression differences appear to be involved in the strain-specific changes of lung mechanic properties.     

Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization

To identify pathways involved in adult lung regeneration, we employ a unilateral pneumonectomy (PNX) model that promotes regenerative alveolarization in the remaining intact lung. We show that PNX stimulates pulmonary capillary endothelial cells (PCECs) to produce angiocrine growth factors that induce proliferation of epithelial progenitor cells supporting alveologenesis. Endothelial cells trigger expansion of cocultured epithelial cells, forming three-dimensional angiospheres reminiscent of alveolar-capillary sacs. After PNX, endothelial-specific inducible genetic ablation of Vegfr2 and Fgfr1 in mice inhibits production of MMP14, impairing alveolarization. MMP14 promotes expansion of epithelial progenitor cells by unmasking cryptic EGF-like ectodomains that activate the EGF receptor (EGFR). Consistent with this, neutralization of MMP14 impairs EGFR-mediated alveolar regeneration, whereas administration of EGF or intravascular transplantation of MMP14(+) PCECs into pneumonectomized Vegfr2/Fgfr1-deficient mice restores alveologenesis and lung inspiratory volume and compliance function. VEGFR2 and FGFR1 activation in PCECs therefore increases MMP14-dependent bioavailability of EGFR ligands to initiate and sustain alveologenesis.     

Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549 alveolar epithelial cells

During lung injury alveolar epithelial cells are directly exposed to changes in PO(2) and PCO(2). Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O(2)-5% CO(2)) or hypercapnia (21% O(2-) 15% CO(2)) and expression of surfactant protein C was measured and compared to normal conditions (21% O(2)- 5% CO(2)). Cell cycle progression and apoptosis were measured by flow cytometric analysis. RESULTS: A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-alpha partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. CONCLUSIONS: Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.     

Upregulation of erythropoietin receptor during postnatal and postpneumonectomy lung growth

Circulating erythropoietin (EPO) stimulates erythrocytosis, whereas organ-specific local EPO receptor (EPOR) expression has been linked to angiogenesis, tissue growth, and development. On the basis of the observation of concurrent enhancement of lung growth and erythrocyte production during exposure to chronic hypoxia, we hypothesized that a paracrine EPO system is involved in mediating lung growth. We analyzed EPOR protein expression in normal dog lung tissue during postnatal maturation and during compensatory lung growth after right pneumonectomy (PNX). Membrane-bound EPOR was significantly more abundant in the immature lung compared with mature lung and in the remaining lung 3 wk after PNX compared with matched sham controls. COOH-terminal cytosolic EPOR peptides, which were even more abundant than membrane-bound EPOR, were also upregulated in immature lung but differentially processed after PNX. Apoptosis was enhanced during both types of lung growth in direct relationship to cellular proliferation and EPOR expression. We conclude that both developmental and compensatory lung growth involve paracrine EPO signaling with parallel upregulation but differential processing of EPOR.     

与成纤维细胞共培养的肺泡II型上皮细胞的生物学特性

建立胎鼠肺泡II型上皮细胞(AECII)与肺成纤维细胞(LF)共培养模型,观察与LF共培养下AECII的生物学特性。倒置相差显微镜观察AECII形态和基本生长情况;RT-PCR和流式细胞术分别检测肺泡表面活性蛋白-C(SP-C)、水通道蛋白5(AQP5)mRNA及蛋白质表达;流式细胞术检测细胞周期及Ki67表达。结果显示,与LF共培养时,AECII能较好地保留其细胞形态,SP-CmRNA及其蛋白质表达明显增加,而AQP5mRNA及其蛋白质表达则明显减少;LF促进AECII增殖,使G2/M、S期细胞及表达Ki67 细胞的比率明显增多。结果提示,AECII与LF共培养时,能更好地保留其细胞形态、分化及增殖特性。     

Preventing mediastinal shift after pneumonectomy impairs regenerative alveolar tissue growth

To examine the effects of mechanical lung strain on regenerative growth of alveolar septal tissue after pneumonectomy (PNX), we replaced the right lungs of adult dogs with a custom-shaped inflatable silicone prosthesis. The prosthesis was either inflated (Inf) to maintain the mediastinum at the midline or deflated to allow mediastinal shift. The animals were euthanized approximately 15 mo later, and the lungs were fixed at a constant distending pressure. With the Inf prostheses, lung expansion, alveolar septal tissue volumes, surface areas, and diffusing capacity of the tissue-plasma barrier were significantly lower than with the deflated prostheses; the expected post-PNX tissue responses were impaired by 30-60%. Capillary blood volume was significantly higher with Inf prostheses, consistent with microvascular congestion. Measurements in the Inf group remained consistently and significantly higher than those expected for a normal left lung, indicating persistence of partial compensation. In one dog, delayed deflation of the prosthesis 9-10 mo after PNX led to vigorous lung expansion and septal tissue growth, particularly of type II epithelial cells. We conclude that mechanical lung strain is a major signal for regenerative lung growth; however, other signals are also implicated, accounting for a significant fraction of the compensatory response to PNX.     

Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage

Cigarette smoking causes apoptotic death, senescence, and impairment of repair functions in lung fibroblasts, which maintain the integrity of alveolar structure by producing extracellular matrix (ECM) proteins. Therefore, recovery of lung fibroblasts from cigarette smoke-induced damage may be crucial in regeneration of emphysematous lung resulting from degradation of ECM proteins and subsequent loss of alveolar cells. Recently, we reported that bone marrow-derived mesenchymal stem cell-conditioned media (MSC-CM) led to angiogenesis and regeneration of lung damaged by cigarette smoke. In this study, to further investigate reparative mechanisms for MSC-CM-mediated lung repair, we attempted to determine whether MSC-CM can recover lung fibroblasts from cigarette smoke-induced damage. In lung fibroblasts exposed to cigarette smoke extract (CSE), MSC-CM, not only inhibited apoptotic death, but also induced cell proliferation and reversed CSE-induced changes in the levels of caspase-3, p53, p21, p27, Akt, and p-Akt. MSC-CM also restored expression of ECM proteins and collagen gel contraction while suppressing CSE-induced expression of cyclooxygenase-2 and microsomal PGE(2) synthase-2. The CSE-opposing effects of MSC-CM on cell fate, expression of ECM proteins, and collagen gel contraction were partially inhibited by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. In rats, MSC-CM administration also resulted in elevation of p-Akt and restored proliferation of lung fibroblasts, which was suppressed by exposure to cigarette smoke. Taken together, these data suggest that MSC-CM may recover lung fibroblasts from cigarette smoke-induced damage, possibly through inhibition of apoptosis, induction of proliferation, and restoration of lung fibroblast repair function, which are mediated in part by the PI3K/Akt pathway.     

Long-term post-pneumonectomy pulmonary adaptation following all-trans-retinoic acid supplementation

In adult dogs following right pneumonectomy (PNX) and receiving all-trans-retinoic acid (RA) supplementation for 4 mo, we found modestly enhanced alveolar-capillary growth in the remaining lung without enhanced resting lung function (J Appl Physiol 96: 1080-1089 and 96: 1090-1096, 2004). Since alveolar remodeling progresses beyond this period and the lipid-soluble RA continues to be released from tissue stores, we hypothesized that RA supplementation may exert additional long-term effects. To examine this issue, adult male litter-matched foxhounds underwent right PNX followed by RA supplementation (2 mg/kg po 4 days/wk, n = 6) or placebo (n = 4) for 4 mo. Cardiopulmonary function was measured at rest and during exercise at 4 and 20 mo post-PNX. The remaining lung was fixed under a constant airway pressure for morphometric analysis. Comparing RA treatment to placebo controls, there were no differences in aerobic capacity, cardiopulmonary function, or lung volume at rest or exercise. Alveolar-capillary basal lamina thickness and mean harmonic thickness of air-blood diffusion barrier were 23-29% higher. The prevalence of double-capillary profiles remained 82% higher. Absolute volumes of septal interstitium, collagen fibers, cells, and matrix were 32% higher; the relative volumes of other septal components and alveolar-capillary surface areas expressed as ratios to control values were up to 24% higher. Thus RA supplementation following right PNX modestly and persistently enhanced long-term alveolar-capillary structural dimensions, especially the deposition of interstitial and connective tissue elements, in such a way that caused a net increase in barrier resistance to diffusion without improving lung mechanics or gas exchange.     

Adenovirus-mediated transfer of the SOCS-1 gene to mouse lung confers protection against hyperoxic acute lung injury

Suppressor of cytokine signaling-1 (SOCS-1) is a member of the suppressor of cytokine signaling family of proteins and an inhibitor of interleukin-6 (IL-6) signaling. SOCS-1 has been shown to protect cells from cellular damage and apoptosis induced by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interferon gamma (IL-γ). However, it is not known whether increased SOCS-1 is protective during pulmonary oxidative stress. Therefore, we hypothesized that increased SOCS-1 in the lungs of mice would be protective in the setting of hyperoxic lung injury. We administered SOCS-1 adenovirus (Ad-SOCS-1) intratracheally into the lungs and exposed the mice to 100% O₂. Mice infected with GFP adenovirus (Ad-GFP) were used as controls. Mice treated with Ad-SOCS-1 had enhanced survival in 100% oxygen compared to Ad-GFP-administered mice. After 3 days of hyperoxia, Ad-GFP mice were ill and tachypnic and died after 4 days. In contrast, all Ad-SOCS-1-treated mice survived for at least 6 days in hyperoxia and 80% survived beyond 7 days. Ad-SOCS-1 transfection protected mouse lungs from injury as indicated by lower lung wet/dry weight, alveolar–capillary protein leakage, reduced infiltration of inflammatory cells, and lower content of thiobarbituric acid-reactive substances in lung homogenate. Our results also indicated that Ad-SOCS-1 significantly inhibits hyperoxia-induced ASK-1 (apoptosis signal-regulating kinase 1) expression. Taken together, these findings show that increased expression of adenovirus-mediated SOCS-1 in the lungs of mice significantly protects against hyperoxic lung injury.     

IL10 deficiency promotes alveolar enlargement and lymphoid dysmorphogenesis in the aged murine lung

The connection between aging‐related immune dysfunction and the lung manifestations of aging is poorly understood. A detailed characterization of the aging IL10‐deficient murine lung, a model of accelerated aging and frailty, reconciles features of both immunosenescence and lung aging in a coherent model. Airspace enlargement developed in the middle‐aged (12 months old) and aged (20–22 months old) IL10‐deficient lung punctuated by an expansion of macrophages and alveolar cell apoptosis. Compared to wild‐type (WT) controls, the IL10‐deficient lungs from young (4‐month‐old) mice showed increased oxidative stress which was enhanced in both genotypes by aging. Active caspase 3 staining was increased in the alveolar epithelial cells of aged WT and mutant lungs but was greater in the IL10‐deficient milieu. Lung macrophages were increased in the aged IL10‐deficient lungs with exuberant expression of MMP12. IL10 treatment of naïve and M2‐polarized bone marrow‐derived WT macrophages reduced MMP12 expression. Conditioned media studies demonstrated the secretome of aged mutant macrophages harbors reduced AECII prosurvival factors, specifically keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), promotes cell death, and reduces survival of primary alveolar epithelial cells. Compared to WT controls, aged IL10‐deficient mice have increased parenchymal lymphoid collections comprised of a reduced number of apoptotic cells and B cells. We establish that IL10 is a key modulator of airspace homeostasis and lymphoid morphogenesis in the aging lung enabling macrophage‐mediated alveolar epithelial cell survival and B‐cell survival within tertiary lymphoid structures.     

Progressive adult-onset emphysema in transgenic mice expressing human MMP-1 in the lung

Mice with lung-specific expression of human matrix metalloproteinase-1 (MMP-1) develop emphysematous changes similar to those seen in smoking-induced emphysema in humans. Morphometric analyses of three transgenic lines [homozygous colony (Col) 34, Col 50, and Col 64] with varying temporal expression of MMP-1 were undertaken to determine the validity of this animal as a model of adult-onset emphysema. Line 50 mice, which have early expression of MMP-1 (14 days postconception), exhibited morphometric changes by 5 days of age. In contrast, homozygous line 34 and 64 with delayed expression (birth and 2 wk of age) were normal up until 4 wk of age when progressive changes in their mean linear intercept were first noted. In contrast, heterozygous mice from line 34 with lower transgene expression did not develop emphysema until 1 yr of age. The changes in mean linear intercept coincided with an increase in lung compliance. Emphysema in these mice was associated with decreased immunostaining for type III collagen within the alveolar septa. This study provides evidence that MMP-1 induces progressive adult-onset emphysema by the selective degradation of type III collagen within the alveolar wall.