In the development of the pulmonary vasculature, both the pulmonary trunk and pulmonary arch arteries receive contributions from cardiac neural crest cells, which migrate from the dorsal neural tube and populate the pharyngeal arches and outflow tract of the developing heart (Kirby and Waldo 1995). Between the 4th and 8th weeks of gestation, concurrent with the start of lung budding, the cardiac outflow tract is septated into the aorta and pulmonary trunk from either the fusion of the distal and proximal outflow cushions of the embryonic heart, the elongation of an aortopulmonary septum, or a combination of both paradigms (Webb et al. 2003). During this process, the aortopulmonary septum can be observed growing in between the fourth and sixth paired pharyngeal arches, which will form components of the mature vasculature: the fourth pharyngeal arches contribute to the aortic arch and subclavian arteries, while the right sixth regresses and the left sixth persists to form the ductus arteriosus and the pulmonary arteries by the 7th week (Poelmann and Gittenberger-de Groot 2005).

The development of the intrapulmonary vessels (i.e., the branching arterial tree most closely associated with the airways and the pulmonary veins running along the periphery of the terminal respiratory units) and the peripheral microvasculature of the lung parenchyma has sparked much recent investigation (de Mello and Reid 2000; de Mello et al. 1997; Hall et al. 2000, 2002; Maeda et al. 2002; Parera et al. 2005; Schachtner et al. 2000; Schwarz et al. 2009). Although several hypotheses have been put forward, two essential processes are implicated: vasculogenesis, which describes the de novo formation of primitive blood plexuses from the aggregation of mesodermal endothelial progenitor cells (angioblasts), and angiogenesis, wherein new vessels are formed by sprouting from existing ones (Poole and Coffin 1989). Intuitively, one might predict that the two processes follow a successive program. For example, in the establishment of the embryonic vascular network, a primitive vascular plexus formed by vasculogenesis is subsequently remodelled and expanded by angiogenesis (Patan 2000). In the development of the pulmonary vasculature, however, the degree of involvement of either process has not been definitively answered, reflecting differences in methodology and interpretation, and possibly the intrinsic morphological differences among and between human samples and animals models.

The earliest comprehensive studies in the mouse and human described the proximal macrovasculature deriving from angiogenic sprouting of the pulmonary vascular trunks, while the distal microvasculature formed by vasculogenesis of hematopoietic cell lakes diffusely coalescing around actively branching distal airway epithelium, starting in the late embryonic phase (de Mello and Reid 2000; de Mello et al. 1997). In this model, the two processes function concurrently but independently, with fusion of the proximal vasculature and peripheral capillary networks occurring relatively late in pulmonary vascular development (E13–14 in the mouse and 10–11 weeks in the human). In contrast, others described the patency between the proximal and distal vessels—and hence a functional pulmonary circulation—occurring much earlier in development (by E10.5 in mice and 34 days gestation in humans, concurrent with the formation of the first airway branches and tracheal separation from the esophagus) (Hall et al. 2000, 2002; Schachtner et al. 2000). This implicated vasculogenesis as the predominate mechanism of the intrapulmonary vascular system, in which endothelial cell precursors (angioblasts) form primary capillary plexuses surrounding developing epithelial buds in the lung periphery. These plexuses continue to expand and coalesce to form arteries and veins more proximal to the terminal buds, suggesting that unlike the development of the airways, which branch and develop centrifugally from the hilum towards the periphery, the primordial vascular system starts from the periphery and expands proximally (Hall et al. 2000, 2002). However, recent reports have suggested that early pulmonary vascular growth and expansion occurs primarily by angiogenesis (Parera et al. 2005; Schwarz et al. 2009). In agreement with previous studies (Hall et al. 2000; Schachtner et al. 2000), vascular circulation connected to the central vessels appears to be in place at the earliest signs of lung development by the observation of primitive erythrocytes in the lumen of plexiform vessels surrounding growing epithelial buds (at E9.5 in mice) (Parera et al. 2005). However, angiogenesis was suggested to occur predominantly because of the description of these newly formed plexuses remodelling into proximal vessels and the absence of putative angioblasts as assessed by immunostaining for vascular endothelial growth factor receptor (VEGFR)-2 and CD34 (early endothelial markers). Hence, rather than distal vasculogenesis, they proposed distal angiogenesis, in which new capillaries arise from existing ones at the lung periphery (Fig. 1.2). Schwarz and coauthors (2009) agree that the expansion of the pulmonary vasculature in mice occurs primarily by angiogenesis, based upon the co-expression of CD31, phosphorylated VEGFR-2, and fluorescent lectin perfusion, which combined indicated the presence of functional vessels by E11.5. They suggest that previous studies which used VEGFR-2 (rather than phosphorylated VEGFR-2) to indicate angioblasts (and hence vasculogenesis) (Schachtner et al. 2000) reflect uncommitted pluripotent cells. Furthermore, they put forth the interesting model that vessel ingrowth begins within the lung mesenchyme and not in close apposition to the epithelial-mesenchymal interface as is commonly described (e.g., Parera et al. (2005)). In this model, new vessels must then expand and extend from the mesenchyme towards the epithelial-mesenchymal junction via angiogenesis beginning at E14.5, likely as a response to vascular endothelial growth factor (VEGF-A) signalling originating from the basilar epithelium. This would suggest that before E14.5, vessels may not necessarily follow or influence airway branching, although diffusible tissue interactions between the vasculature and epithelium are still required for normal growth and maintenance of both, which is consistent with the degeneration of either tissue layer when cultured in isolation (Gebb and Shannon 2000; Shannon et al. 1998, 1999). Indeed, while chemical inhibition of VEGFR-2 signalling halted vascular growth in E11.5 mouse explants, epithelial branching was only modestly attenuated (Groenman et al. 2007). Hence, disruption of this vessel extension to the primordial parenchyma by inhibiting VEGF-A signalling would prevent the progression of epithelial-endothelial reciprocal signalling and thus the blockage of further lung morphogenesis beyond E14.5, as observed (van Tuyl et al. 2005; Yamamoto et al. 2007).

Normal lung function requires the development of an air-blood barrier of maximal surface area and minimal thinness. This is achieved by the formation of the alveoli beginning in late prenatal human development (week 36) and continuing through early postnatal life in a process termed “alveolarization” (also referred to as “alveologenesis”) (Fig. 1.3). Although birth is often considered the end of organogenesis and the start of growth and maturation, the development and maturation of alveoli in humans and most other mammals is largely, and often exclusively, a postnatal process, ceasing only several years after birth in humans (Burri 1997). The end result of alveolarization and isometric parenchymal growth produces a lung with alveolar and capillary surface areas at least 20 times that of the neonate (Zeltner et al. 1987).

Alveolarization occurs as the immature saccules which form the lung parenchyma are subdivided into smaller units by the formation and subsequent extension of secondary septa (singular, “septum”). This is followed by a period of microvascular maturation, in which the double capillary layer lining the alveoli is remodelled into a monolayer by capillary fusion and intussusceptive growth. The seminal reports which introduced this model of alveolarization proposed that the two processes acted somewhat independently of each other: secondary septation was proposed to occur mostly between postnatal days (pnd) 4–14 in the rat, followed successively by microvascular maturation primarily during the third postnatal week (Burri 1974). Hence, the formation of alveoli by further septation was thought impossible following the completion of microvascular maturation, which placed the endpoint of alveolarization in rats and humans conservatively at 3 weeks and 2–3 years postnatal, respectively (Zeltner et al. 1987).

The determination of the endpoint of alveolar development is of much clinical relevance since a longer period of alveolarization increases the window of opportunity for morphological disruptions to occur. For example, alveolar development could arrest without subsequent “catch-up” as a consequence of acute lung disease experienced early in life or from iatrogenic interventions, such as from glucocorticoid therapy in the treatment of asthma. Again, while the postnatal gas-exchange surface area dramatically increases with respect to longitudinal growth (Zeltner et al. 1987), this could arise solely from the isometric growth of the lung parenchyma rather than from the formation of new alveolar septa.

In fact, two recent stereological studies in mice and rats from one group have reported that isometric growth of the lung can only account for a modest increase in septal surface area during postnatal development (Mund et al. 2008; Schittny et al. 2008). By their estimates, the formation of alveoli occurs well beyond the stage of microvascular maturation, ceasing only in young adulthood in mice (pnd 36) and rats (pnd 60). These authors propose that alveolarization proceeds in two phases: “classical” alveolarization occurs prior to microvascular maturation (pnd 4–21) by the formation of secondary septa from immature (capillary bilayer) septa, which accounts for 40 and 50 % of septa present in the adult rat and mouse, respectively, and a second “late” phase (pnd 21–adulthood), in which alveolar septa must arise from mature (capillary monolayer) septa, accounting for 50 and 40 % of septa in the adult rat and mouse, respectively (the remaining 10 % of septa appear to be formed prenatally from branching morphogenesis) (Mund et al. 2008; Schittny et al. 2008). Importantly, the second phase appears to require localized angiogenesis at the base of the newly forming septum, since the capillary microvasculature has already thinned to a monolayer (Mund et al. 2008; Schittny et al. 2008). These results are in agreement with a similar stereological study in rhesus monkeys, which also reported alveolarization continuing through to the 8th postnatal year (Hyde et al. 2007), suggesting that the same may well be true in humans.

While similar analyses in humans are for practical reasons not possible, measurements of pulmonary diffusing capacity for carbon monoxide (DL_CO, used indirectly as a measurement of parenchymal surface area) and alveolar volume (V _A) in infants aged 2–23 months showed that the DL_CO/V _A ratio remained constant, suggesting that lung growth occurred primarily by alveolar septation within the first 2 years of life (Balinotti et al. 2009). In contrast, DL_CO/V _A decreased with increasing age in children ages 6–17 years, consistent with isometric parenchymal lung growth outpacing alveolar septation (Stam et al. 1996). However this does not definitively answer the question of when alveolar development in humans is completed. Indeed, the size of the terminal airspaces in mice and rats was observed to change over the course of postnatal development as a result of the changing rates of septal formation and parenchymal growth, which vary over maturation, although with some interspecies variability (Mund et al. 2008; Schittny et al. 2008). Hence, whether alveolarization in humans principally occurs en masse in a relatively short period of time (so-called bulk alveolarization), or more gradually, and perhaps variably, alongside longitudinal growth, has yet to be determined.

BPD is a condition which affects nearly one third of neonates with birth weights less than 1,000 g (Walsh et al. 2006). The aetiology of the disease is multifactorial and develops in response to lung injury experienced from both environmental factors (hyperoxia relative to in utero, infection, nutritional deficits, lung immaturity) and strategies designed to manage premature infant care (mechanical ventilation, antenatal steroid administration). Genetic susceptibility, such as with respect to surfactant synthesis and inflammatory response, is also being recognized as a significant contributing factor (Abman et al. 2008).

BPD is presently characterized by an arrest in alveolar septation and dysmorphic vascular growth, leading to a reduction in gas-exchange surface area and respiratory potential (Bourbon et al. 2005). Similarly, gas exchange is impaired in adult-onset emphysema, which arises from damage to preexisting alveoli (Morris and Sheppard 2006). Studies which identify the fundamental regulators which orchestrate normal alveolarization and how these pathways are disrupted in conditions such as BPD and emphysema are critical for the development of therapies which can treat these debilitating diseases. However, the mechanisms involved in the various morphogenetic processes of alveolarization are not well understood. Multiple stimuli, including growth and transcription factors, extracellular matrix (ECM) proteins, hormones, and oxygen exposure coordinated through autocrine and paracrine interactions between the epithelium, fibroblast derivatives, and the ECM, work together in concert to sculpt the alveolar architecture (Roth-Kleiner and Post 2005). Conversely, dysregulation of these interactions results in arrested alveolar development. Next we provide a short updated review of factors known to be essential for normal alveolarization and which have largely been uncovered in the context of chronic lung diseases, such as BPD.