Developmental alveologenesis: new roles for ApoE and LDL receptor.

Pulmonary developmental alveologenesis occurs, in substantial part, by subdivision (septation) of the gas-exchange saccules of the morphologically immature lung. It determines the starting point of age- and disease-related alveolar loss. Because alveologenesis requires additional cell membranes, we previously asked whether apoE-/-, which delivers lipids to cells, affects pulmonary alveologenesis; male apoE-/- mice had impaired alveologenesis. We now report that, in contrast to male apoE-/- mice, female apoE mice had full developmental alveologenesis. Among mice null for LDL receptor (Ldlr-/-), the receptor for apoE-/-, females had full alveologenesis; by contrast, Ldlr-/- males, as previously shown for apoE males, had impaired alveologenesis. Thus, the absence of apoE and its receptor, Ldlr, results in impaired developmental alveologenesis in males, but their absence does not impair architectural developmental alveologenesis in females. We conclude 1) regulation of alveologenesis is a new function for apoE and Ldlr, 2) one expressed in a sexually dimorphic manner, and 3) females have different molecular requirements for alveologenesis than males, which protects them from its impairment by the absence of apoE and its receptor.

Apoetm1Unc mice have impaired alveologenesis, low lung function, and rapid loss of lung function.

Diminished lung function, indicated by a low forced expiratory volume in one second (FEV1), and short physical stature, predict early mortality from all causes, including cardiovascular, among smokers and never smokers. The basis for these associations is unclear, and, it is not known if there is a pulmonary morphological component to the relationship between low FEV1 and early death in a general population. Some apolipoprotein E genotypes also predict atherosclerosis and early mortality. These considerations led us to examine the Apoe(tm1Unc) (Apoe) mouse, in which the apolipoprotein E gene is deleted, and that develops dyslipidemia, atherosclerosis at an early age, and has a shorter life span than the founder wild-type (wt) strain. We asked if Apoe mice have a morphological or functional pulmonary phenotype. We measured the size, number, and surface area of pulmonary gas-exchange units (alveoli) and mechanical properties of the lung. Compared with wt mice, Apoe mice had: 1) diminished developmental alveologenesis, 2) increased airway resistance in early adulthood, 3) high lung volume and high dynamic and static compliance in later adulthood, 4) more rapid loss of lung recoil with age, and 5) were less long than wt mice. These findings in mice indicate the association of a low FEV1 with early death in humans may have developmental, and accelerated ageing, related pulmonary components, and that dietary, genetic, or dietary and genetic influences, on lipid metabolism may be an upstream cause of inflammation and oxidative stress, currently considered to be major risk factors for COPD.

Estrogen receptor-alpha regulates pulmonary alveolar loss and regeneration in female mice: morphometric and gene expression studies.

Pulmonary alveoli, especially in females, are estrogen-responsive structures: ovariectomy in wild-type (WT) adult mice results in alveolar loss, and estradiol replacement induces alveolar regeneration. Furthermore, estrogen receptor (ER)-alpha and ER-beta are required for the developmental formation of a full complement of alveoli in female mice. We now show ovariectomy resulted in alveolar loss in adult ER-beta(-/-) mice but not in adult ER-alpha(-/-) mice. Estradiol treatment of ovariectomized ER-beta(-/-) mice induced alveolar regeneration. In ovariectomized WT mice, estradiol treatment resulted, within 1 h, in RNA-level gene expression supportive of processes needed to form an alveolar septum, e.g., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion. Among these processes, protein expression supporting angiogenesis and cell replication was elevated 1 and 3 h, respectively, after estradiol treatment; similar findings were not present in either mutant. We conclude: 1) loss of signaling via ER-beta is not required for postovariectomy-induced alveolar loss or estradiol-induced regeneration; this indicates ER-alpha is key for estrogen-related alveolar loss and regeneration in adult female mice; 2) taken together with prior work showing that developmental formation of a full complement of alveoli requires ER-alpha and ER-beta, the present findings indicate the developmental and regenerative formation of alveoli are regulated differently, i.e., signaling for alveolar regeneration is not merely a recapitulation of signaling for developmental alveologenesis; and 3) the timing of estradiol-induced gene expression in lung supportive of processes required to form a septum differs between ovariectomized WT and ER-beta(-/-) mice.

Rapid onset of gene expression in lung, supportive of formation of alveolar septa, induced by refeeding mice after calorie restriction.

Alveolar regenerative gene expression is unidentified partly because its onset, after a regenerative stimulus, is unknown. Toward addressing this void, we used a mouse model in which calorie restriction produces alveolar loss, and ad libitum access to food after calorie restriction induces alveolar regeneration. We selected four processes (cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion) that would be required to convert a flat segment of alveolar wall into a septum that increases gas-exchange surface area. Global gene expression supportive of processes required to form a septum was present within 3 h of allowing calorie-restricted mice food ad libitum. One hour after providing calorie-restricted mice food ad libitum, RNA-level expression supportive of cell replication was present with little evidence of expression supportive of angiogenesis, extracellular matrix remodeling, or guided cell motion. Cell replication was more directly assayed by measuring DNA synthesis in lung. This measurement was made 3 h after allowing calorie-restricted mice food ad libitum because translation may be delayed. Ad libitum food intake, following calorie restriction, elevated DNA synthesis. Thus RNA expression 1 h after allowing calorie-restricted mice food ad libitum supported increased cell replication; measurements at 3 h revealed increased DNA synthesis and RNA expression, supportive of the three other processes required to form a septum. These findings identify the first hour after providing calorie-restricted mice ad libitum access to food as the onset of gene expression in this model that supports processes needed for alveolar regeneration.

Toward therapeutic pulmonary alveolar regeneration in humans.

In humans, age results in loss of pulmonary alveoli; menopause accelerates loss of diffusing capacity, an index of alveolar surface area; and disease (e.g., chronic obstructive pulmonary disease) results in loss of alveoli. Thus, an important goal for investigators is to generate knowledge that allows induction of pulmonary alveolar regeneration in humans. Our enthusiasm for this goal and our assessment of its feasibility are based on work in several laboratories over the last decade that has disproved the notion that pulmonary alveoli are incapable of regeneration, and on the growing evidence that signals that regulate programs of alveolar turnover (loss and regeneration) are conserved from rodents to humans. We review animal models of alveolar loss and regeneration and their conservation during evolution, and hence their relevance to humans.

Estrogen receptor regulation of pulmonary alveolar dimensions: alveolar sexual dimorphism in mice.

Female rats and mice have smaller and, per body mass (BM), more alveoli and alveolar surface area (Sa) than males of their respective species. This sexual dimorphism becomes apparent about the time of sexual maturity. It is prevented in rats (not tested in mice) by ovariectomy at age 3 wk. In female mice, estrogen receptor (ER)-alpha and ER-beta are required for formation of alveoli of appropriate size and number. We now report the average volume of an alveolus (va) and the number of alveoli per body mass (Na/BM) were not statistically different between ER-alpha(-/-) and wild type (wt) males. However, the combination of a larger value for va and a smaller value for Na/BM, though neither parameter achieved a statistically significant intergroup difference, resulted in a statistically significant lower Sa/BM in ER-alpha(-/-) males compared with wt males. In ER-beta(-/-) males, va was bigger and Na/BM and Sa/BM were lower compared with wt males. Wt males had larger alveoli and lower Na/BM and Sa/BM than wt females. The wt sexual dimorphism of va, Na/BM, and Sa/BM was absent in ER-alpha(-/-) mice. Alveolar size did not differ between ER-beta(-/-) females and males but Na/BM and Sa/BM were greater in ER-beta(-/-) females than in ER-beta(-/-) males. The results in male mice, with prior findings in female mice, 1) demonstrate estrogen receptors have a smaller effect on alveolar dimensions in male than female mice, 2) show ER-alpha and ER-beta are required for the sexual dimorphism of alveolar size, and 3) show ER-alpha is needed for the sexual dimorphism of body mass-specific alveolar number and surface area.

Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice.

Lung tissue elastic recoil and the dimension and number of pulmonary gas-exchange units (alveoli) are major determinants of gas-exchange function. Loss of gas-exchange function accelerates after menopause in the healthy aged and is progressively lost in individuals with chronic obstructive pulmonary disease (COPD). The latter, a disease of midlife and later, though more common in men than in women, is a disease to which women smokers and never smokers may be more susceptible than men; it is characterized by diminished lung tissue elastic recoil and presently irremediable alveolar loss. Ovariectomy in sexually immature rats diminishes the formation of alveoli, and estrogen prevents the diminution. In the present work, we found that estrogen receptor-alpha and estrogen receptor-beta, the only recognized mammalian estrogen receptors, are required for the formation of a full complement of alveoli in female mice. However, only the absence of estrogen receptor-beta diminishes lung elastic tissue recoil. Furthermore, ovariectomy in adult mice results, within 3 wk, in loss of alveoli and of alveolar surface area without a change of lung volume. Estrogen replacement, after alveolar loss, induces alveolar regeneration, reversing the architectural effects of ovariectomy. These studies 1) reveal estrogen receptors regulate alveolar size and number in a nonredundant manner, 2) show estrogen is required for maintenance of already formed alveoli and induces alveolar regeneration after their loss in adult ovariectomized mice, and 3) offer the possibility estrogen can slow alveolar loss and induce alveolar regeneration in women with COPD.

Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice.

A technically easy, noninvasive means of delivering molecules to alveoli, which act selectively or specifically in the lung, would be experimentally and therapeutically useful. As proof of principle, we took advantage of the spreading ability of pulmonary surface active material (InfaSurf), mixed it with elastase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) small inhibitory RNA (siRNA), or all-trans retinoic acid (ATRA), and instilled microliter amounts of the mixture into the nose of lightly anesthetized mice. One instillation of elastase caused diffuse alveolar destruction (emphysema) demonstrating widespread alveolar delivery. A single nasal instillation of GAPDH siRNA, compared with scrambled GAPDH siRNA, lowered GAPDH protein in lung, heart, and kidney by approximately 50-70% 1 and 7 days later. To test the possibility of lung-specific delivery of a potentially therapeutic drug, we administered ATRA and monitored its effect on expression of cellular retinol binding protein (CRBP)-1 mRNA, whose translation product is a key molecule in retinoid metabolism. Given intranasally, ATRA elevated CRBP-1 mRNA 4.3-fold in a lung-specific manner. The same dose and dose schedule of ATRA given intraperitoneally increased CRBP-1 mRNA only approximately 1.8-fold in lung; intraperitoneally administered ATRA elevated expression of CRBP-1 mRNA 1.7-fold or more in brain cortex, cerebellum, and testes, thereby increasing the risk of untoward effects. This simple noninvasive technique allows regulation of specific proteins in the lung and lung-specific delivery of reagents of experimental and potentially therapeutic importance.

Expression of lung uncoupling protein-2 mRNA is modulated developmentally and by caloric intake.

Lung expresses a high concentration of uncoupling protein-2 (UCP-2) mRNA, but neither its pulmonary regulation nor function is known. We measured lung UCP-2 mRNA expression in two animal models: in neonatal rats when both the metabolic rate, as measured by oxygen consumption, and levels of serum free fatty acids (FFAs) increase and in adult mice during decreased food intake, when levels of serum FFAs increase but the metabolic rate decreases. In rat lung, the concentration of UCP-2 mRNA was low and unchanged during late gestation, increased approximately twofold within 6 hrs after birth, and, compared with late gestation, remained approximately threefold higher from day 1 to adulthood. The early postnatal rise in the lung UCP-2 mRNA concentration was partially blocked by an antithyroid drug and was increased by treatment with triiodothyronine. Unlike lung, heart UCP-2 mRNA levels were lower during adulthood than at day 15. In adult mice, lung UCP-2 mRNA concentrations increased approximately fivefold within 12 hrs of 67% calorie restriction (CR), remained elevated during 2 weeks of CR, fell to control levels within 24 hrs of refeeding (CR-RF), and positively correlated with serum FFA concentrations. Heart UCP-2 expression during CR and CR-RF was similar to that of lung; liver UCP-2 mRNA levels were slightly lower during CR and returned to control levels during CR-RF. These data suggest that the regulation of UCP-2 is at least partly tissue-specific and that, in the adult mouse, lung UCP-2 is regulated not by oxygen consumption but by FFAs. Moreover, lung UCP-2 mRNA levels in mice fed ad libitum was increased by the intraperitoneal administration of Intralipid, a 20% fat emulsion. On the basis of these data in adult mice, together with the findings of others that levels of FFAs increase by 2 hrs after birth, we propose lung UCP-2 is regulated by FFA.

DNA microarray analysis of neonatal mouse lung connects regulation of KDR with dexamethasone-induced inhibition of alveolar formation.

Treatment of newborn mice with dexamethasone (Dex) inhibits the subdivision of lung saccules to form alveoli; treatment with all-trans retinoic acid (RA) prevents this inhibition of septation. To better understand the early molecular signals responsible for the effects of Dex and RA, Affymetrix gene profiling was done on RNA isolated from 4-day-old mice after treatment with 1) diluent, 2) RA (1 mg/kg), 3) Dex (0.7 microg/pup), or 4) RA + Dex. Each sample was assayed in duplicate on U74Av2 GeneChips. Data were analyzed with Affymetrix suite 5.0, corrected for saturation, and evaluated with GeneSpring 5.1 software. Stringent filtering of data by the global error model and condition-to-condition comparisons was used to identify 46 genes demonstrating significantly different expression between the lungs of Dex- and RA + Dex-treated mice. A query of the gene ontology database revealed that the major biological processes affected by treatment with Dex and RA were cell growth/maintenance and cellular communication. On the basis of microarray data analysis, we hypothesize that Dex-induced inhibition of septation is associated with a block in angiogenesis due to downregulation of the kinase domain receptor (KDR), also known as VEGF receptor-2 and fetal liver kinase, and that the downregulation of KDR is prevented by treatment with RA.

Calorie-related rapid onset of alveolar loss, regeneration, and changes in mouse lung gene expression.

Calorie restriction, followed by ad libitum refeeding, results, respectively, in loss and regeneration of pulmonary alveoli. We now show 35% of alveoli are lost within 72 h of onset of calorie restriction ((2/3) decreased daily chow intake), and an additional 12% of alveoli are lost over a subsequent 12 days of calorie restriction. Tissue necrosis was not seen. Within 72 h of refeeding, after 15 days of calorie restriction, the number of alveoli returns to precalorie restriction values. Microarray lung gene profiling, in conjunction with Western and RNase protection assay, demonstrate an increase of granzyme and caspase gene expression 2-3 h after onset of calorie restriction. By 12 h, granzyme and caspase expression is no longer increased, but tumor necrosis factor death receptor expression is elevated. At 336 h, Fas death receptor expression is increased. Because granzymes are found only in cytotoxic lymphocytes (CTLs) and natural killer (NK) cells, we suggest calorie restriction activates these cells, initiating a series of molecular events that results in alveolar destruction. The evidence of involvement of CTLs and NK cells and the absence of necrosis are similar to alveolar destruction in chronic obstructive pulmonary disease.

Lung retinol storing cells synthesize and secrete retinoic acid, an inducer of alveolus formation.

Retinoids play a key role in the formation of pulmonary alveoli. Lipid interstitial cells (LICs) of the alveolar wall store retinol and are concentrated at sites of alveolus formation, suggesting they are an endogenous source of retinoids for alveolus formation. We show in cultured rat lung cells that LICs synthesize and secrete all-trans retinoic acid (ATRA); its secretion is halved by dexamethasone, an inhibitor of alveolus formation. In a second alveolar wall cell, the pulmonary microvascular endothelial cell (PMVC), ATRA increases expression of the mRNA of cellular retinol binding protein-I (CRBP-I), a protein involved in ATRA synthesis. Serum-free, exogenous ATRA-free medium conditioned by LICs rich in retinol storage granules caused a 10-fold greater increase of CRBP-I mRNA in PMVCs than media conditioned by LICs with few retinol storage granules. This action of medium conditioned by retinol storage granule-rich LICs is decreased by a retinoic acid receptor pan-antagonist and by a retinoid X receptor pan-antagonist, suggesting the responsible molecule(s) is a retinoid and that retinoid signaling occurs in a paracrine fashion.

Retinoic acid receptor-alpha regulates pulmonary alveolus formation in mice after, but not during, perinatal period.

The formation of pulmonary alveoli in mice and rats by subdivision of alveolar saccules that constitute the newborn's gas-exchange region ends by approximately postnatal day 14. However, alveoli continue to form after age 14 days until age approximately 40 days by means other than septation of the saccules present at birth. With the use of morphometric procedures and retinoic acid receptor (RAR)-alpha+/+ and RAR-alpha-/- mice, we now show the volume of individual alveoli (va), the number of alveoli (Na), and alveolar surface area (Sa) are the same in 14-day-old RAR-alpha+/+ and RAR-alpha-/- mice. However, at age 50 days, va is larger, and Na and Sa are smaller, in RAR-alpha-/- than in RAR-alpha+/+ mice, although total lung volume is the same in both groups. These findings, and prior data showing RAR-beta is an endogenous inhibitor of alveolus formation during, but not after, the perinatal period, indicate there are developmental period-specific regulators of alveolus formation and that total lung volume and alveolar dimensions may have different regulators.

Lung alveoli: endogenous programmed destruction and regeneration.

Mammalian alveoli, complex architectural and cellular units with dimensions that are linked to the organism's O2 consumption (VO2), are thought to be destroyed only by disease and not to spontaneously regenerate. Calorie restriction of adult mammals lowers VO2, and ad libitum refeeding returns VO2 to pre-calorie-restriction values. We took advantage of these relationships and tested the hypothesis in adult mice that calorie restriction (two-thirds reduction for 2 wk) followed by ad libitum refeeding (3 wk) would cause alveolar destruction and regeneration, respectively. Calorie restriction diminished alveolar number 55% and alveolar surface area 25%. Refeeding fully reversed these changes. Neither manipulation altered lung volume. Within 72 h, calorie restriction increased alveolar wall cell apoptosis and diminished lung DNA (approximately 20%). By 72 h of refeeding, alveolar wall cell replication increased and lung DNA rose to amounts in mice that were never calorie restricted. We conclude that adult mice have endogenous programs to destroy and regenerate alveoli, thereby raising the danger of inappropriate activation but the possibility of therapeutic induction, if similar programs exist in humans.