Retinoic acid-induced alveolar cellular growth does not improve function after right pneumonectomy.

To determine whether all-trans retinoic acid (RA) treatment enhances lung function during compensatory lung growth in fully mature animals, adult male dogs (n = 4) received 2 mg x kg(-1) x day(-1) po RA 4 days/wk beginning the day after right pneumonectomy (R-PNX, 55-58% resection). Litter-matched male R-PNX controls (n = 4) received placebo. After 3 mo, transpulmonary pressure (TPP)-lung volume relationship, diffusing capacities for carbon monoxide and nitric oxide, cardiac output, and septal volume (V(tiss-RB)) were measured under anesthesia by a rebreathing technique at two lung volumes. Lung air and tissue volumes (V(air-CT) and V(tiss-CT)) were also measured from high-resolution computerized tomographic (CT) scans at a constant TPP. In RA-treated dogs compared with controls, TPP-lung volume relationships were similar. Diffusing capacities for carbon monoxide and nitric oxide were significantly impaired at a lower lung volume but similar at a high lung volume. Whereas V(tiss-RB) was significantly lower at both lung volumes in RA-treated animals, V(air-CT) and V(tiss-CT) were not different between groups; results suggest uneven distribution of ventilation consistent with distortion of alveolar geometry and/or altered small airway function induced by RA. We conclude that RA does not improve resting pulmonary function during the early months after R-PNX despite histological evidence of its action in enhancing alveolar cellular growth in the remaining lung.     

Retinoic acid induces nonuniform alveolar septal growth after right pneumonectomy.

To determine whether all-trans retinoic acid (RA) enhances compensatory lung growth in fully mature animals, adult male dogs (n = 4) received 2 mg x kg(-1) x day(-1) po RA 4 days/wk beginning the day after right pneumonectomy (R-PNX, 55-58% resection). Litter-matched male R-PNX controls (n = 4) received placebo. After 4 mo, the remaining lung was fixed by tracheal instillation of fixatives at a constant airway pressure for detailed morphometric analysis. After RA treatment compared with placebo, lung volume was slightly but not significantly lower. Volume density of septum to lung was 37% higher because of a 50 and 25% higher volume density of capillary and septal tissue, respectively. Mean septal thickness was 27% higher. Absolute volumes of endothelial cells and capillary blood were 31-37% higher, whereas epithelial and interstitial volumes were not different between groups. Absolute alveolar-capillary surface areas did not differ between groups, and alveolar septal surface-to-volume ratio was 20% lower in RA-treated animals. RA treatment exaggerated interlobar differences in morphometric indexes and caused alveolar capillary morphology to revert to a more immature state. Thus RA treatment during early post-R-PNX adaptation preferentially enhanced alveolar capillary and endothelial cell volumes consistent with formation of new capillaries, but the associated septal distortion precluded a corresponding increase in gas-exchange surface or morphometric estimates of lung diffusing capacity.     

Adaptation of respiratory muscle perfusion during exercise to chronically elevated ventilatory work.

Pneumonectomy (PNX) leads to chronic asymmetric ventilatory loading of respiratory muscles (RM). We measured RM energy requirements during exercise from RM blood flow (Q) using a fluorescent microsphere technique in dogs that had undergone right PNX as adults (adult R-PNX) or as puppies (puppy R-PNX), compared with dogs subjected to right thoracotomy without PNX as puppies (Sham) and to left PNX as adults (adult L-PNX). Ventilatory work (W) was measured during exercise. RM weight was determined post mortem. After adult and puppy R-PNX, the right hemidiaphragm becomes grossly distorted, but W and right costal muscle mass increased only after adult R-PNX. After adult L-PNX, the diaphragm was undistorted; W and left hemidiaphragm RM Q were elevated, but muscle mass did not increase. Mass of parasternal muscle did not increase after adult R-PNX, despite increased Q. Thus muscle mass increased only in response to the combination of chronic stretch and dynamic loading. There was a dorsal-to-ventral gradient of increasing Q within the diaphragm, but the distribution was unaffected by anatomic distortion, hypertrophy, or workload, suggesting a fixed pattern of neural activation. The diaphragm and parasternals were the primary muscles compensating for the asymmetric loading from PNX.     

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.     

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.     

Inhibition of compensatory lung growth in endothelial nitric oxide synthase-deficient mice

Pneumonectomy results in rapid compensatory growth of the remaining lung and also leads to increased flow and shear stress, which are known to stimulate endothelial nitric oxide synthase (eNOS). Nitric oxide is an essential mediator of vascular endothelial growth factor-induced angiogenesis, which should necessarily occur during compensatory lung growth. Thus our hypothesis is that eNOS is critical for compensatory lung growth. To test this, left pneumonectomy was performed in eNOS-deficient mice (eNOS-/-), and compensatory growth of the right lung was characterized throughout 14 days postpneumonectomy and compared with wild-type pneumonectomy and sham controls. Compensatory lung growth was severely impaired in eNOS-/- mice, as demonstrated by significant reductions in lung weight index, lung volume index, and volume of respiratory region. Also, pneumonectomy-induced increases in alveolar surface density and cell proliferation were prevented in eNOS-/- mice, indicating that eNOS plays a role in alveolar hyperplasia. Compensatory lung growth was also impaired in wild-type mice treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester. Together, these results indicate that eNOS is critical for compensatory lung growth.     

Lung volumes after an increase in lung distension in pneumonectomized ferrets

To investigate the role of lung distension in compensatory lung growth, the right lung of each of 21 adult male ferrets was replaced with a silicone rubber balloon filled with mineral oil. Three to thirteen weeks after surgery, the oil was removed through a subcutaneous port. Lung volumes were measured serially until 3-6 wk after balloon deflation. With pneumonectomy the total lung capacity (TLC) decreased to less than 50% of the preoperative value and remained essentially unchanged while the balloon was inflated. At balloon deflation, TLC and vital capacity did not change immediately, whereas functional residual capacity increased by 44%, indicating a change of 2-3 cmH2O in end-expiratory transpulmonary pressure. TLC increased by 10% within 3 days and continued to increase over the subsequent 3-5 wk by a total of 25% over TLC at balloon deflation. There was little difference in this response between animals whose balloons were deflated 3 wk after surgery and those in which deflation was delayed up to 13 wk. After pneumonectomy in the adult ferret, the remaining lung increases in volume in response to an increase in lung distension even weeks or months after surgery. The extent to which this volume increase involves lung tissue growth or depends on previous lung resection is at present unknown. This model may be useful for studies of the mechanisms by which lung distension influences lung volume and compensatory lung growth.     

Preventing mediastinal shift after pneumonectomy does not abolish physiological compensation.

To determine the role of mediastinal shift after pneumonectomy (PNX) on compensatory responses, we performed right PNX in adult dogs and replaced the resected lung with a custom-shaped inflatable silicone prosthesis. Prosthesis was inflated (Inf) to prevent mediastinal shift, or deflated (Def), allowing mediastinal shift to occur. Thoracic, lung air, and tissue volumes were measured by computerized tomography scan. Lung diffusing capacities for carbon monoxide (DL(CO)) and its components, membrane diffusing capacity for carbon monoxide (Dm(CO)) and capillary blood volume (Vc), were measured at rest and during exercise by a rebreathing technique. In the Inf group, lung air volume was significantly smaller than in Def group; however, the lung became elongated and expanded by 20% via caudal displacement of the left hemidiaphragm. Consequently, rib cage volume was similar, but total thoracic volume was higher in the Inf group. Extravascular septal tissue volume was not different between groups. At a given pulmonary blood flow, DL(CO) and Dm(CO) were significantly lower in the Inf group, but Vc was similar. In one dog, delayed mediastinal shift occurred 9 mo after PNX; both lung volume and DL(CO) progressively increased over the subsequent 3 mo. We conclude that preventing mediastinal shift after PNX impairs recruitment of diffusing capacity but does not abolish expansion of the remaining lung or the compensatory increase in extravascular septal tissue volume.     

Partial pneumonectomy of telomerase null mice carrying shortened telomeres initiates cell growth arrest resulting in a limited compensatory growth response

Telomerase mutations and significantly shortened chromosomal telomeres have recently been implicated in human lung pathologies. Natural telomere shortening is an inevitable consequence of aging, which is also a risk factor for development of lung disease. However, the impact of shortened telomeres and telomerase dysfunction on the ability of lung cells to respond to significant challenge is still largely unknown. We have previously shown that lungs of late generation, telomerase null B6.Cg-Terc(tm1Rdp) mice feature alveolar simplification and chronic stress signaling at baseline, a phenocopy of aged lung. To determine the role telomerase plays when the lung is challenged, B6.Cg-Terc(tm1Rdp) mice carrying shortened telomeres and wild-type controls were subjected to partial pneumonectomy. We found that telomerase activity was strongly induced in alveolar epithelial type 2 cells (AEC2) of the remaining lung immediately following surgery. Eighty-six percent of wild-type animals survived the procedure and exhibited a burst of early compensatory growth marked by upregulation of proliferation, stress response, and DNA repair pathways in AEC2. In B6.Cg-Terc(tm1Rdp) mice carrying shortened telomeres, response to pneumonectomy was characterized by decreased survival, diminished compensatory lung growth, attenuated distal lung progenitor cell response, persistent DNA damage, and cell growth arrest. Overall, survival correlated strongly with telomere length. We conclude that functional telomerase and properly maintained telomeres play key roles in both long-term survival and the early phase of compensatory lung growth following partial pneumonectomy.     

Vascular growth and remodeling in compensatory lung growth following right lobectomy.

Studies in animal models have shown that, following lobectomy (LBX), there is compensatory growth in the remaining lung. The vascular growth response following right LBX (R-LBX) is poorly understood. To test the hypothesis that arterial growth and remodeling occur in response to LBX, in proportion to the amount of right lung tissue removed, two (24% of lung mass; R-LBX2 group) or three right lobes (52% of lung mass; R-LBX3 group) were removed via thoracotomy from adult rats. Sham control animals underwent thoracotomy only. Arteriograms were generated 3 wk after surgery. The areas of the left lung arteriogram, arterial branching, length of arterial branches, arterial density, and arterial-to-alveolar ratios were measured. To determine whether R-LBX causes vascular remodeling and pulmonary hypertension, muscularization of arterioles and right ventricular hypertrophy were assessed. Lung weight and volume indexes were greater in R-LBX3. Arterial area of the left lung increased 26% in R-LBX2 and 47% in R-LBX3. The length of large arteries increased in R-LBX3 and to a lesser extent in R-LBX2. The ratio of distal pulmonary arteries to alveoli was similar after R-LBX2 compared with sham but was 30% lower in R-LBX3. Muscularization of arterioles increased after R-LBX3, but not in R-LBX2. Right ventricular hypertrophy increased 50-70% in R-LBX3, but not in R-LBX2. Whereas removal of three right lung lobes induced arterial growth in the left lungs of adult rats, which was proportionate to the number of lobes removed, the ratio of distal pulmonary arteries to alveoli was not normal, and vascular remodeling and pulmonary hypertension developed.     

Recruitment of lung diffusing capacity with exercise before and after pneumonectomy in dogs

Although the left lung constitutes 42% of the total by weight and volume in dogs, carbon monoxide diffusing capacity (DL) after left pneumonectomy in adults falls less than 30% at rest, indicating a significant increase of DL in the remaining lung. DL normally increases during exercise, presumably by recruitment of alveolar capillaries and surface area as lung volume (Vs) and pulmonary blood flow (Qc) increase. We asked whether the increase of DL in the remaining lung after pneumonectomy in adult dogs could be explained by this kind of passive recruitment by the increased volume and Qc in the remaining lung. We measured the relationship between DL and Qc with a rebreathing technique at increasing treadmill loads in adult foxhounds, before and 6 mo after left pneumonectomy, and the relationship between DL and Vs by the same technique under anesthesia as Vs was expanded. DL was reduced by 29.1% at rest and 26.5% with heavy exercise after left pneumonectomy, indicating either recruitment or new growth in the right lung. With the assumption that the right lung normally receives 58% of the Qc and contains 58% of the DL, DL of the right lung increased with Qc in accordance with the following relationships before and after left pneumonectomy: right lung DL (before pneumonectomy) = 6.44 + 2.40(Qc) (r = 0.963) and right lung DL (after pneumonectomy) = 7.51 + 1.75(Qc) (r = 0.958). Only approximately 7% of the increase in DL from rest to peak exercise could be attributed to the increase in Vs during exercise before pneumonectomy and approximately 15% after pneumonectomy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regenerative growth of respiratory bronchioles in dogs

Loss of lung units due to pneumonectomy stimulates growth of the remaining lung. It is generally believed that regenerative lung growth involves only alveoli but not airways, a dissociated response termed "dysanaptic growth." We examined the structural response of respiratory bronchioles in immature dogs raised to maturity after right pneumonectomy. In another group of adult dogs, we also examined the effect of preventing mediastinal shift after right pneumonectomy on the response of respiratory bronchioles. In immature dogs after pneumonectomy, the volume of the remaining lung increased twofold, with no change in volume density, numerical density, or mean diameter of respiratory bronchiole, compared with that in the control lung. The number of respiratory bronchiole segments and branch points increased proportionally with lung volume. In adult dogs after pneumonectomy, prevention of mediastinal shift reduced lung strain at a given airway pressure, but lung expansion and regenerative growth of respiratory bronchiole were not eliminated. We conclude that postpneumonectomy lung growth is associated with proliferation of intra-acinar airways. The proportional growth of acinar airways and alveoli should optimize gas exchange of the regenerated lung by enhancing gas conductance and mixing efficiency within the acinus.     

Inhibition of compensatory renal growth by indomethacin

Renal prostaglandins may be important in the modulation of compensatory renal growth. Reductions in renal mass are associated with increased synthesis of these substances by the remaining kidney, and inhibition of prostaglandin synthesis diminishes renal function in partially nephrectomized animals and in patients with reduced functioning renal mass. We examined the effects of uninephrectomy and treatment with indomethacin on renal prostaglandin E2 and 6-keto prostaglandin F1 alpha concentrations in adult male Sprague Dawley rats. The renal content of these prostaglandins was significantly increased in the remaining kidney two days following uninephrectomy (p less than 0.01). Treatment with 5 mg/kg/day of indomethacin over this period abolished the compensatory increase in renal prostaglandin synthesis and significantly attenuated compensatory increases in renal mass, protein and RNA concentrations (p less than 0.05). No alterations in kidney weight, protein or RNA concentrations were found in intact animals treated with the same dose of indomethacin. These findings suggest renal prostaglandins may participate in the biological events leading to compensatory renal growth.     

Progressive adaptation in regional parenchyma mechanics following extensive lung resection assessed by functional computed tomography

In adult canines following major lung resection, the remaining lobes expand asymmetrically, associated with alveolar tissue regrowth, remodeling, and progressive functional compensation over many months. To permit noninvasive longitudinal assessment of regional growth and function, we performed serial high-resolution computed tomography (HRCT) on six male dogs (～9 mo old, 25.0 ± 4.5 kg, ±SD) at 15 and 30 cmH(2)O transpulmonary pressure (Ptp) before resection (PRE) and 3 and 15 mo postresection (POST3 and POST15, respectively) of 65-70% of lung units. At POST3, lobar air volume increased 83-148% and tissue (including microvascular blood) volume 120-234% above PRE values without further changes at POST15. Lobar-specific compliance (Cs) increased 52-137% from PRE to POST3 and 28-79% from POST3 to POST15. Inflation-related parenchyma strain and shear were estimated by detailed registration of corresponding anatomical features at each Ptp. Within each lobe, regional displacement was most pronounced at the caudal region, whereas strain was pronounced in the periphery. Regional three-dimensional strain magnitudes increased heterogeneously from PRE to POST3, with further medial-lateral increases from POST3 to POST15. Lobar principal strains (PSs) were unchanged or modestly elevated postresection; changes in lobar maximum PS correlated inversely with changes in lobar air and tissue volumes. Lobar shear distortion increased in coronal and transverse planes at POST3 without further changes thereafter. These results establish a novel use of functional HRCT to map heterogeneous regional deformation during compensatory lung growth and illustrate a stimulus-response feedback loop whereby postresection mechanical stress initiates differential lobar regrowth and sustained remodeling, which in turn, relieves parenchyma stress and strain, resulting in progressive increases in lobar Cs and a delayed increase in whole lung Cs.     

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.     

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.