Regulation of Alveolarization

Introduction

Lung development is a continuous process, starting very early in embryonic life with the differentiation of the tracheal bud from the ventral side of the primitive gut and ending in postnatal life with the multiplication of alveoli and the maturation of the pulmonary microvasculature. Alveoli represent the functional unit of the lung, the site at which oxygen and carbon dioxide are exchanged between inspired air and the blood. Alveolarization is controlled by many factors, whose expression is tightly regulated temporally and spatially. In humans, the most illustrative pathology of impaired alveolar multiplication is bronchopulmonary dysplasia (BPD), the most common chronic respiratory disease in premature infants, and a source of significant respiratory morbidity. Moderate and severe forms of this disease are characterized by prolonged respiratory insufficiency, with persistent requirement for supplemental oxygen beyond 36 weeks of postmenstrual age. Impaired alveolarization, with alveolar hypoplasia and altered microvascular maturation, are characteristic pathophysiologic features of BPD.

Timing of Alveolar Multiplication During Lung Development

The first distal air spaces are represented by primitive saccules during the saccular stage. The alveolar stage succeeds the saccular stage and is characterized by a dramatic increase in the gas exchange surface due to the subdivision of the primitive saccules by new interair-space walls resulting in new alveoli. Formation of alveoli may be pre- or postnatal, depending on the species. It is almost entirely prenatal in species such as guinea pigs or lambs. In contrast, in rodents and humans, the formation of alveoli is essentially postnatal. Therefore, rodents are most often used as a model of human alveolar development. The duration of alveolar multiplication during postnatal life has been the subject of many studies. Most recent studies confirm that new septa are formed until young adulthood. Using a direct and unbiased assessment of the number of alveoli in the rat model, Tschanz and associates demonstrated a biphasic behavior of alveolarization. In this model, the early phase of alveolarization corresponds to a very high production rate of alveoli, frequently described as bulk alveolarization . During this initial phase, the number of alveoli increased in rats from less than 1 million at postnatal day 4, to approximately 3.5 million at day 10, and to over 14 million at day 21. This represents an impressive rate of 800,000 new alveoli per day. During this initial period, the volumetric expansion of the alveolar air space does not follow, and therefore the alveoli are becoming much smaller. This first phase is followed by a second phase from day 21 to day 60, corresponding to young adulthood, with a slower rate of alveolar multiplication, and the formation of an additional 5 million alveoli. During this second phase, the formation of new alveoli is less important than the growth of the alveoli, leading to an increase in the mean airspace volume. Recent stereologic-based longitudinal study reported dynamic changes in structural changes during postnatal mouse lung development. Alveologenesis was clearly evident over the early postnatal phase. The saccular or alveolar density was multiplied threefold between the 5th and 7th postnatal day, and fivefold between the 5th and 10th postnatal day. Alveolar density peaked at the 39th postnatal day and remained unchanged at 9 months but was reduced by 22 months. Stereologic analysis revealed a progressive decrease in the mean saccular or alveolar volume of the lung over the first 10 days of life. Mean septal wall thickness dramatically decreased over the first 10 days of postnatal life, with a twofold reduction between the 5th and 10th postnatal day, and continued to decrease to the 28th postnatal day. These experimental data were confirmed in humans. Based on human lung tissues obtained by autopsy, Herring and associates demonstrated that the number of alveoli in the human lung increased exponentially during the first 2 years of life but continued to increase, albeit at a reduced rate, through adolescence. The estimated number of alveoli for the whole human lung is around 100 × 10 ⁶ alveoli by 1 month of postnatal age, and reaches over 500 × 10 ⁶ alveoli by 15 years of age.

Structural Changes During the Alveolar Stage

Primitive saccules are delineated by thick intersaccular walls, or primary septa . The alveolar stage is characterized by the formation of numerous small ridges from the saccular wall, called secondary septa, which grow in a centripetal manner into the saccules to subdivide them into alveoli. Among the variety of factors that participate in the control of budding of secondary septa, spatial and temporal changes in extracellular elastin and laminin distribution appeared as critical. Elastin deposition in the thickness of primary septa appears to have a spatially instructive role, as sites of elastic fiber formation correspond precisely to the location of future buds. However, 3-D analysis recently showed that the isolated patches in the tips of the “finger-like” protrusions were not actually isolated but were parts of continuous elastin fibers that rim the alveolar opening. The growth of secondary septa into the lumen of the terminal saccule is accompanied by migration and proliferation of fibroblasts. Lipofibroblasts (LFs) and myofibroblasts (MFs) are two lineages of mesenchymal cells with fibroblast characteristics that are identified during alveolarization. LFs contain lipid vacuoles and are located at the base of newly formed septa, in close proximity to the alveolar type II cells (AECII) and endothelial cells. In the rat, the quantity of LFs doubles between P4 and P7, with an accompanying increase in cellular lipid content. LFs may constitute a stem-cell niche for AECII stem cells. MFs are nonlipid-containing, α-smooth muscle actin–positive interstitial cells and are located adjacent to collagen and elastic fibers. MFs may be derived from platelet-derived growth factor receptor-α (PDGFRA)–positive cells and play a key role in elastogenesis. Elastin fibers are arranged in an orderly and predictable manner within the alveolar septa. Crosslinking of elastin monomers is a critical process in alveolarization, and inhibition of this process impairs secondary septation (see below). Elastin fibers are in close proximity with collagen fibers, suggesting that both fibers are mechanically interconnected. Increased deposition of elastin and collagen fibers does not drive maturational changes in lung tissue mechanics. Indeed, changes in tissue viscoelastic properties with maturation are determined mainly by other components of extracellular matrix, such as glycosaminoglycans and proteoglycans, which are dynamically controlled during postnatal stages of pulmonary development. At the beginning of the alveolar stage, both primary and secondary septa show a double capillary layer, as one capillary is associated with each surface of the forming alveolar septa. In a recent study that imaged newborn mouse lung with serial block-face scanning electron microscopy, new 3D information was provided on the structure of the alveolar capillary network in the newborn lung. In particular, the so-called double-layered capillary network of developing lungs was identified more as a single network extending in all three dimensions rather than two (nearly separated) networks within one septum.

Along with the emergence of secondary septa, a progressive thinning of interstitial tissue is observed. It is likely that this decrease in interstitial volume allows both capillary networks to come into close contact and to potentially fuse, so that a single capillary system forms in the alveolar wall. It was postulated that the formation of secondary septa can continue as long as a double capillary network is present and that the end of alveolarization occurs in parallel with capillary fusion. At the end of the bulk alveolarization in rats, a 20% to 25% fraction of the capillary network is still immature, thus potentially explaining the continued alveolarization beyond this period. ^, At 60 days in rodents, 5% to 10% of the capillary network remains immature and might contribute to alveolar regeneration in adults. Furthermore, local reduplication of the capillary network was also described as the basis of newly forming septa in adult rodents.

The period of alveolization is accompanied by the differentiation of alveolar epithelial cells, which is covered in another chapter of this book.

Regulators of the Normal Alveolar Multiplication

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