Trophic factors in the neonatal gastrointestinal tract

Introduction

Gastrointestinal growth and function are significant facets of neonatal development that are highly regulated by both intrinsic and extrinsic factors. The development and function of gastrointestinal organs during the neonatal period have been extensively examined using novel molecular biological and genetic approaches, including the use of in vitro enteroids and organoids cultivated from fetal and neonatal tissues. This has revealed microenvironments within intestinal crypts and villi, allowing stem cell proliferation in crypts while simultaneously regulating cellular differentiation, function, and eventual quiescence at the villus tip. Extrinsic factors, such as nutrients and hormones, are also important determinants that prepare the fetus and neonate gut for birth and weaning. Enteral nutrition is the most significant stimulus for gastrointestinal growth and protein synthesis. Select nutrients, such as glutamine, arginine, threonine, and leucine; nucleotides; and short-chain fatty acids (SCFAs) and long-chain fatty acids (LCFAs) also have trophic effects. The gut is also a significant endocrine organ secreting hormones such as glucagon-like peptide 2 that have profound effects on gastrointestinal growth. Other hormones, such as glucocorticoids, are important in triggering fetal intestinal maturation before birth. Additionally, local and endocrine growth factors such as serotonin and the epidermal growth factor and insulin-like growth factor (IGF) families play significant roles in the regulation of gastrointestinal function and proliferation. There is a highly coordinated balance between cellular proliferation and function in the gastrointestinal tract that is imperative for neonatal health and survival.

The nature of gut growth

The neonatal period is a highly dynamic period of gastrointestinal growth and functional development. In the case of the intestine, this includes the development of swallowing and mature motility patterns, tissue vascular hemodynamics, and nutrient transporters. Together, these physiologic changes during the fetal-neonatal period facilitate the transition from placental nutrient assimilation to oral ingestion via the gastrointestinal tract. Intestinal epithelial growth at the tissue and cellular levels is characterized by increased cell numbers (i.e., hyperplasia ) and increased cellular size (i.e., hypertrophy ). Intestinal growth also involves expansion of the number and size of crypt and villus units. An important aspect of growth in the gut is the continual proliferation, migration, and loss of epithelial cells along the mucosal surface. In the intestine, this process involves multiple cell lineages (absorptive enterocyte, goblet, Paneth, endocrine, tuft) that differentiate from pluripotent stem cells located in the crypt. Growth is also characterized by structural and functional changes in innervation and vascularization within the gut. Gastrointestinal growth involves the proliferation, growth, and development of cells and structures, including blood vessels, endothelial cells, smooth muscle cells, submucosal nerves, and myenteric nerves. The normal growth and development of the gastrointestinal tract is critical to normal development of the neonate, as the gut is a central organ for nutrient digestion and absorption and is a major environmental interface for innate immune function, nutrient sensing, and neuroendocrine crosstalk between the gut and brain. The timing and characteristics of neonatal gastrointestinal growth are exquisitely coordinated with the events of birth and weaning to ensure survival of the organism. The regulation of neonatal gastrointestinal growth is complex and involves multiple and often redundant factors. Among these factors are intrinsic cell programs or signals arising from gene expression, as well as extracellular signals such as peptide growth factors, hormones, nutrients, and microbes, which originate from surrounding cells, the blood, and the gut lumen.

In the past decade, the development of in vitro models of fetal and neonatal gastrointestinal physiology in the form of intestinal enteroids and organoids has provided new insights about the role of stem cells and growth factors in the proliferation and maintenance of the intestinal epithelium. Intestinal organoids are specifically representative of fetal tissue and have been used to examine signaling pathways that determine the eventual developmental fate (endoderm, mesoderm, or ectoderm) of pluripotent stem cells, from gastrulation onward. Growth factors such as TGF-β initiate gastrulation in early fetal life, but other factors such as Wnt, fibroblast growth factors (FGFs), retinoic acid, and bone morphogenetic protein (BMP) pathways are essential for the development and subsequent differentiation of the intestinal epithelium ( Fig. 5.1 ). Enteroids are cultured from stem cells present in the base of intestinal crypts and represent the physiology of the tissue the cells were isolated from. For example, if stem cells are harvested from diseased or aging tissue, they will differentiate into enteroids that reflect the ratio of epithelial cell lineages observed in the original tissue. In enteroid culture, Wnt signaling is amplified by R-spondin to maximize enteroid proliferation and survivability, while epidermal growth factor (EGF) has a similar, profound mitogenic effect on stem cells. In intestinal tissue, BMP signaling is greatest at the villus tip and lowest at the crypt base, driving terminal differentiation. Additionally, R-spondin has been shown to promote the expansion of the stem cell niche in intestinal tissue. Other growth factors used in organoid and enteroid culture are still under investigation for their clinical significance in nonneoplastic and neonatal gut tissue.

Gut adaptation in the perinatal period

Normal gastrointestinal growth and development during fetal life are critical to facilitate successful adaptation from nutritional support via umbilical circulation to oral ingestion of breast milk. An increase in circulating fetal glucocorticoid concentration just prior to and during vaginal birth is an important trigger of gut functional development. ^, In the neonatal period, growth of the gastrointestinal tract is influenced by multiple physiologic factors that serve to prepare the developing neonate for separation from maternal nutritional support (i.e., weaning). In addition, several important environmental cues signal adaptive changes in gastrointestinal function to facilitate postweaning survival. For example, the microbial colonization of the gut may serve to prime intestinal lymphoid cell development for normal innate and adaptive immune function. ^, During these processes, extracellular signals such as peptide growth factors are believed to be important trophic factors that influence growth. However, the term trophic also pertains to nutrition, and in the case of the gut, nutrients present in amniotic fluid and breast milk are a major trophic influence. Thus there are numerous extracellular trophic signals including foods, nutrients, peptide growth factors, gut peptide hormones, steroid and thyroid hormones, microbes, and neural inputs. The cells within the fetal and neonatal gastrointestinal tract are influenced by extracellular signals from multiple sources, including (1) blood-borne factors in the circulation such as hormones that act via endocrine mechanisms; (2) luminal factors derived from amniotic fluid, mammary secretions, or microbes; and (3) local factors secreted via autocrine or paracrine mechanisms from surrounding cells. Additionally, intrinsic intracellular signals such as transcription factors interact with these extracellular signals to affect mucosal hyperplasia and hypertrophy.

Preterm birth creates major problems for newborn infants’ adaptation to normal oral feeding and enteral nutrition. These problems are often manifested clinically as feeding intolerance, sepsis, and necrotizing enterocolitis (NEC). These problems are linked with immature gastroduodenal motor function, hemodynamic regulation, nutrient malabsorption, a dysfunctional mucosal immune system, and microbial dysbiosis. Thus the challenge for clinicians caring for preterm infants is to provide the appropriate combination of clinical support, nutritional or otherwise, to stimulate the normal growth and development of these organ systems and physiologic functions.

How soon and how much to feed enterally

Enteral nutrition is the most potent trophic stimulus of gastrointestinal tract growth. Enteral feeding acts directly by supplying nutrients for growth and oxidative metabolism of the mucosal epithelial cells. However, it also acts indirectly by triggering the release of local growth factors and gut hormones and activating neural pathways ( Fig. 5.2 ). Neonatal starvation and total parenteral nutrition (TPN) reduce gut tissue mass and mucosal surface area, increase catabolism, and decrease protein synthesis. ^, Evidence from numerous animal studies and some human studies shows that enteral nutrition is critical to maintain normal intestinal growth and development. The enteral nutrient stimulation of gut growth begins in the late-gestation fetus with the onset of amniotic fluid swallowing. Studies in fetal sheep and pigs have shown that preventing amniotic fluid swallowing by esophageal ligation suppresses intestinal growth. ^,

In most infants, enteral feeding of human milk or formula after birth stimulates the growth and adaptive development of the gastrointestinal tract. However, in the immediate postnatal period premature infants normally receive most of their nutrition parenterally due to poor feeding tolerance. Studies in neonatal pigs show that TPN leads to significantly reduced growth and to atrophy of the intestinal mucosa marked by reduced cell proliferation, villus height, and protein synthesis as well as increased apoptosis. Additional studies with neonatal animals and human infants suggest that lack of enteral nutrition is also associated with reduced secretion of many gut peptide hormones and growth factors as well as dysbiotic intestinal microbiota colonization, all of which may be linked to reduced gut functional development during TPN.

Therefore, in premature infants, the question is how soon and how much to feed enterally. Several clinical studies have demonstrated that the practice of minimal enteral feeding or trophic feeding can enhance gastrointestinal motility and intestinal function and improve feeding outcomes ( Fig. 5.1 ). The introduction of minimal enteral nutrition usually starts at birth at a daily intake of ~24–25 mL/kg, an amount that is insufficient for the total nutrient needs of the infant. Meta-analyses have shown that while minimal enteral nutrition reduces time to full feeding and length of hospital stay, the impact on overall health and disease risk for premature infants is yet to be established. ^, However, recent clinical trials suggest that advancement of enteral feeding by 20–30 mL/kg/d may be safe and may not increase the risk of NEC. ^{, ,} Clinical studies in premature infants have indicated that enteral feeding is positively linked to intestinal mucosal growth but that lactose digestion is more closely correlated to lactase activity and gestational age than feeding status. ^, Studies in piglets suggest that enteral nutrition is critical to maintain normal lactose digestion and glucose absorption. Studies in neonatal piglets also suggest that an enteral intake of at least 40% of total nutrient intake is necessary to maintain normal growth, which would imply that minimal enteral nutrition may not be trophic to gut mucosa. An additional consideration is whether to provide enteral nutrition intragastrically, intraduodenally, as a bolus, or continuously. Evidence from clinical studies is equivocal as to clinical outcome. ^, Studies in piglets suggest that, in comparison with continuous feeding, bolus feeding resulted in increased gut growth and improvement in trophic insulin response in skeletal muscle in term pigs ^, but was not linked to secretion of trophic gut peptides or improvements in insulin response to feeding in preterm pigs. ^,

The trophic role of breast milk versus formula

The relative significance of milk-borne trophic factors has been an intensely studied area of pediatric nutrition and gastroenterology. Several trophic peptide growth factors are present in mother’s own breast milk but not in infant formulas and have been implicated in the beneficial outcomes of breast-fed infants, particularly in the reduced incidence of NEC and sepsis. However, the stability of these growth factors in donor human milk that has been pasteurized has not been well established. Studies conducted in neonatal animals have shown evidence that breast milk has a greater trophic effect on the gastrointestinal tract than formula, as measured by typical indices of structural and cellular growth. However, the most significant advantage of breast milk on the neonatal intestine, especially of preterm infants, may be related not to growth but to mucosal barrier and immune function. There is considerable evidence showing that immunoprotective factors in breast milk (e.g., secretory IgA, lactoferrin, oligosaccharides) act to modulate mucosal immune function and bacterial colonization, thereby limiting the incidence of infection, sepsis, and NEC. Other novel human milk, plasma, and bovine colostrum–derived bioactive ingredients are being explored for their utility to reduce bacteria-mucosal interaction and therefore improve mucosal growth by reducing inflammation. Many of the trophic factors in milk are polypeptides that survive digestion, retain their biological activity, and interact with specific receptors present on the mucosal epithelium of neonates. Several studies have shown that these milk-borne growth factors stimulate neonatal intestinal growth when given in purified and recombinant forms either orally or systemically. Moreover, in preterm neonates the presence of increased intestinal permeability could facilitate the intestinal absorption of milk-borne peptide growth factors; however, there are limited instances where this process has been found to be physiologically significant.

Another important stimulus of intestinal growth associated with feeding is the secretion of bile acids from the liver. Historically, bile acids were solely considered as detergent molecules that emulsify lipids and function in fat digestion and absorption. In the past two decades, the scope of bile acid biology has greatly expanded, with studies showing that luminal bile acids act as signaling molecules that regulate gastrointestinal functions by binding to a host of membrane and nuclear receptors present on epithelial cells as well as other cell types located within the small intestine and colon. Secondary bile acids present in the colon can directly stimulate epithelial proliferation by activating epithelial growth factor receptor pathways. Studies in neonatal piglets have shown that enterally administering chenodeoxycholic acid was able to prevent TPN-induced small intestinal atrophy and restore normal glucagon-like peptide 2 (GLP-2) secretion. ^, In the distal ileum, bile acids act as ligands for farnesoid X receptor (FXR), triggering the production of fibroblast growth factor 19 (FGF19). FGF19 has multiple systemic effects, but the primary target is to reduce bile acid synthesis in the liver by reducing the expression of CYP7a1. Additionally, FGF19 has been shown to have an insulin-like effect on hepatic protein synthesis and gluconeogenesis, along with a proliferative effect on hepatocytes via the receptors FGFR4 and βklotho. However, the direct effect of FGF19 on intestinal epithelial function remains to be determined.