Wei Hsueh

Neonatal Necrotizing Enterocolitis: Clinical Considerations and Pathogenetic Concepts

Necrotizing enterocolitis (NEC), a disease affecting predominantly premature infants, is a leading cause of morbidity and mortality in neonatal intensive care units. Although several predisposing factors have been identified, such as prematurity, enteral feeding, and infection, its pathogenesis remains elusive. In the past 20 years, we have established several animal models of NEC in rats and found several endogenous mediators, especially platelet-activating factor (PAF), which may play a pivotal role in NEC. Injection of PAF induces intestinal necrosis, and PAF antagonists prevent the bowel injury induced by bacterial endotoxin, hypoxia, or challenge with tumor necrosis factor-a (TNF) plus endotoxin in adult rats. The same is true for lesions induced by hypoxia and enteral feeding in neonatal animals. Human patients with NEC show high levels of PAF and decreased plasma PAF-acetylhydrolase, the enzyme degrading PAF. The initial event in our experimental models of NEC is probably polymorphonuclear leukocyte (PMN) activation and adhesion to venules in the intestine, which initiates a local inflammatory reaction involving proinflammatory mediators including TNF, complement, prostaglandins, and leukotriene C4. Subsequent norepinephrine release and mesenteric vasoconstriction result in splanchnic ischemia and reperfusion. Bacterial products (e.g., endotoxin) enter the intestinal tissue during local mucosal barrier breakdown, and endotoxin synergizes with PAF to amplify the inflammation. Reactive oxygen species produced by the activated leukocytes and by intestinal epithelial xanthine oxidase may be the final pathway for tissue injury. Protective mechanisms include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase, and indigenous probiotics such as Bifidobacteria infantis. The former maintains intestinal perfusion and the integrity of the mucosal barrier, and the latter keep virulent bacteria in check. The development of tissue injury depends on the balance between injurious and protective mechanisms.

Role of platelet activating factor and tumor necrosis factor-alpha in neonatal necrotizing enterocolitis

Because previous investigations have suggested that platelet activating factor and tumor necrosis factor-alpha (TNF-alpha) are important mediators of experimental necrotizing enterocolitis in the rat, we measured platelet activating factor, acetylhydrolase (the platelet activating factor breakdown enzyme), and TNF-alpha in the plasma of 12 human neonates with necrotizing enterocolitis and eight age-matched control subjects with similar gestational ages, postnatal ages, and weights. Almost all patients with necrotizing enterocolitis had elevated plasma platelet activating factor values (18.1 +/- 3.6 ng/ml vs. 3.1 +/- 0.9 ng/ml in control subjects, p less than 0.01). Plasma acetylhydrolase activity was lower in patients than in control subjects (10.6 +/- 0.7 nmol/ml/min vs 23.0 +/- 1.4 nmol/ml/min, p less than 0.01). Plasma TNF-alpha concentration was significantly elevated in patients with necrotizing enterocolitis (136 +/- 75 U/ml vs 1.5 +/- 0.8 U/ml, p less than 0.05), although the individual variation was high. There was no correlation between individual TNF-alpha and platelet activating factor levels. We conclude that platelet activating factor and TNF-alpha are elevated in patients with necrotizing enterocolitis and that suppressed platelet activating factor degradation contributes to the increased platelet activating factor levels; platelet activating factor and TNF-alpha may contribute to the pathophysiology of necrotizing enterocolitis.

Role of Asphyxia and Feeding in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a common gastrointestinal disorder affecting premature infants. To investigate critically the importance of the purported risk factors of NEC (formula feeding, asphyxia, bacteria, and prematurity), we developed a neonatal rat model that closely mimics the human disease. Full-term and premature newborn rats were stressed with formula feeding, asphyxia, and/or exogenous bacterial colonization and subsequently evaluated grossly and histologically for the development of intestinal injury. We found that most animals treated with asphyxia, formula feeding, and bacteria developed NEC (77%) and died (86%) by 96 h. All maternally fed animals treated with asphyxia and bacterial colonization survived and had normal intestinal histology. Furthermore, asphyxia was a critical instigating factor, because formula and bacterial exposure without asphyxia resulted in normal intestine and minimal mortality (12%). Enteral bacterial colonization was not a significant determinant of NEC in this model. We conclude that the neonatal rat model is an excellent test system for the study of NEC. As in the human disease, asphyxia and formula feeding play an important role in the pathophysiology of experimental NEC.

Milk fat globule–EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium

Milk fat globule-EGF factor 8 (MFG-E8)/lactadherin participates in several cell surface-mediated regulatory events. Although its mRNA is present in the gut, the physiological roles of MFG-E8 in the intestinal mucosa have not been explored. Here we show that MFG-E8 was expressed in intestinal lamina propria macrophages from mice. Using a wound-healing assay, MFG-E8 was shown to promote the migration of intestinal epithelial cells through a PKCepsilon-dependent mechanism. MFG-E8 bound to phosphatidylserine and triggered reorientation of the actin cytoskeleton in intestinal epithelial cells at the wound edge. Depleting MFG-E8 in mice by administration of anti-MFG-E8 antibody or targeted deletion of the MFG-E8 gene resulted in a slowing of enterocyte migration along the crypt-villus axis and focal mucosal injury. Moreover, in septic mice, intestinal MFG-E8 expression was downregulated, which correlated with intestinal injury, interrupted enterocyte migration, and impaired restitution. Treatment with recombinant MFG-E8 restored enterocyte migration, whereas deletion of MFG-E8 impeded mucosal healing in mice with sepsis. These results suggest that a decrease in intestinal MFG-E8 impairs intestinal mucosal repair in sepsis. Together, our data indicate that MFG-E8 plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing and suggest that recombinant MFG-E8 may be beneficial for the treatment of bowel injuries.

Platelet-activating factor: an endogenous mediator for bowel necrosis in endotoxemia.

We have developed a model of isochemic bowel necrosis in the rat by injecting platelet‐activating factor (PAF) or PAF in combination with bacterial endotoxin. PAF causes profound hypotension, and it has been suggested that it is released during endotoxin shock. Because ischemic bowel necrosis is often associated with shock or infection, it is possible that PAF is the endogenous mediator that causes shock and bowel necrosis during sepsis. In this study, we have demonstrated that: 1) normal intestine contained a small amount of PAF; 2) necrotic lesions of the intestine could be induced by endotoxin injection; 3) PAF production in the bowel is markedly increased in animals treated with endotoxin; 4) pretreatment of the animal with PAF antagonists prevent endotoxin‐induced necrosis; 5) isolated, buffer‐perfused small intestine produced a small quantity of PAF in response to endotoxin injection. Therefore, we conclude that PAF is a likely endogenous mediator in endotoxemia, which causes bowel necrosis and shock.—Hsueh, W.; González‐Crussi, F.; Arroyave, J. L. Platelet‐activating factor: an endogenous mediator for bowel necrosis in endotoxemia. FASEB J. 1: 403‐405; 1987.

Avian influenza receptor expression in H5N1-infected and noninfected human tissues

Avian and human influenza viruses preferentially bind to α‐2,3‐linked and α‐2,6‐linked sialic acids, respectively. Until today, the distributions of these two receptor types had never been investigated in H5N1‐infected human tissue samples. Here, the expression of avian (AIV‐Rs) and human influenza receptors (HuIV‐Rs) is studied in various organs (upper and lower respiratory tracts, brain, placenta, liver, kidney, heart, intestines, and spleen) of two H5N1 cases and 14 control cases. Histochemical stains using biotinylated Maackia amurensis lectin II and Sambucas nigra aggluti‐nin were performed to localize AIV‐Rs and HuIV‐Rs, respectively. Immunohistochemical stainings were performed to identify the receptor‐bearing cells. AIV‐Rs were detected on type II pneumocytes; a limited number of epithelial cells of the upper respiratory tract; and the bronchi, bronchioli, and trachea; as well as on Kupffer cells, glomerular cells, splenic T cells, and neurons in the brain and intestines. HuIV‐Rs were abundantly present in the respiratory tract and lungs. They were also detected on Hofbauer cells, glomerular cells, splenic B cells, and in the liver. Moreover, endo‐thelial cells of all organs examined expressed both receptor types. In conclusion, the distribution pattern of AIV‐Rs is partially inconsistent with the pattern of infected cells as detected in previous studies, which suggests there may be other receptors or mechanisms involved in H5N1 infection. In addition, the diffuse presence of receptors on endothelial cells may account for the multiple organ involvement in H5N1 influenza. Finally, the relative lack of AIV‐Rs in the upper airway may be a one of the factors preventing efficient human‐to‐human transmission of H5N1 influenza.—Yao, L., Korteweg, C., Hsueh, W., Gu, J. Avian influenza receptor expression in H5N1‐infected and noninfected human tissues. FASEB J. 22, 733–740 (2008)

Serum PAF acetylhydrolase increases during neonatal maturation

Acetylhydrolase is an acid-labile, 43 kd protein that catalyzes the degradation of platelet activating factor (PAF), a potent phospholipid inflammatory mediator, to its biologically inactive metabolite lysoPAF. PAF has a short half-life, thus acetylhydrolase plays an important role in its regulation. Since previous work suggests that PAF may be involved in certain neonatal diseases such as necrotizing enterocolitis, we studied the effect of age on acetylhydrolase activity. Serum acetylhydrolase activity was quantified using radio-labelled PAF and measuring reaction products. Serum samples were obtained prospectively from 70 subjects ranging in age from 4 hr to 48 yr. Acetylhydrolase activity was lower for newborns (less than 3 wk) than all other age ranges (8.2 +/- 1.4 nmole/ml/min vs 30.0 +/- 1.6 nmole/ml/min, p less than .01). Furthermore, enzyme activity increased linearly with respect to the natural logarithm of age from 0 days to 6 weeks (r = 0.65, p less than .001). By 6 weeks of life acetylhydrolase activity approached values of older children and adults. Newborn acetylhydrolase activity was similar between term and preterm infants (8.6 +/- 1.9 nmole/ml/min vs 7.2 +/- 2.4 nmole/ml/min, p = NS). We conclude that acetylhydrolase activity is low in human neonates and increases during the first 6 weeks of life. These results suggest that newborn infants may be at increased risk for pathophysiologic processes mediated by PAF.

Hypoxia Causes Ischemic Bowel Necrosis in Rats: The Role of Platelet-Activating Factor (PAF-Acether)

We have previously shown that injection of platelet-activating factor causes necrotizing enterocolitis in the rat and that platelet-activating factor is an endogenous mediator in lipopolysaccharide-induced bowel necrosis. Because hypoxia is a known predisposing factor for neonatal necrotizing enterocolitis, we investigated the effect of hypoxia on platelet-activating factor formation and intestinal necrosis. Young male Sprague-Dawley rats were made severely hypoxic by placing them in a 100% N2 chamber for 2 minutes; moderate hypoxia was accomplished using 10% O2 for 15 or 30 minutes. To evaluate the role of platelet-activating factor on intestinal perfusion and injury, two platelet-activating factor antagonists, SRI 63-441 and WEB 2086, were injected 10 minutes before the hypoxic exposure. We found that plasma platelet-activating factor levels were significantly elevated after 2 minutes of severe hypoxia (13.8 +/- 2.9 ng/mL vs. control 2.1 +/- 0.8 ng/mL) and after 30 minutes of moderate hypoxia (41.1 +/- 11.7 ng/mL). This increase in platelet-activating factor level was not caused by decreased degradation, because neither plasma nor intestinal platelet-activating factor acetylhydrolase was decreased in the hypoxic rats. (Intestinal acetylhydrolase activity was actually increased). Intestinal perfusion was markedly decreased at 30 minutes in hypoxic animals. In contrast, all platelet-activating factor antagonist-treated animals had normal intestinal perfusion. Histological examination of affected bowel from hypoxic animals showed early intestinal necrosis which was completely prevented by pretreatment with SRI 63-441 and WEB 2086. Because 30 minutes of hypoxia also resulted in metabolic acidosis, we further investigated if acidosis alone could induce platelet-activating factor release and bowel injury. We found that acidosis alone resulted in moderate increase of plasma platelet-activating factor but did not produce bowel injury. We conclude that platelet-activating factor plays a central role in mediating hypoxia-induced intestinal necrosis. Acidosis may enhance the effect of hypoxia on platelet-activating factor production.

Necrotizing enterocolitis : role of platelet activating factor, endotoxin, and tumor necrosis factor

Although many risk factors for necrotizing enterocolitis have been described, the pathophysiology remains poorly understood. We developed a model of ischemic bowel necrosis in the rat, similar to human neonatal NEC, by injecting platelet activating factor into the splanchnic circulation. In this review, we describe the role of PAF in shock and bowel injury, discuss some other mediators that may interact with PAF and contribute to the intestinal abnormality, and present evidence associating PAF and human NEC.

Endogenous Nitric Oxide Protects against Platelet-Activating Factor-Induced Bowel Injury in the Rat

ABSTRACT: Platelet-activating factor (PAF) causes bowel necrosis in animal models that is histologically identical to that seen in neonatal necrotizing enterocolitis, but little is known about endogenous mechanisms that might protect against PAF-induced bowel injury. We hypothesized that endogenous nitric oxide might represent such a protective mechanism. Adult male Sprague-Dawley rats were pretreated with 2.5 mg/kg NG-nitro-L-arginine methyl ester (L-NAME), a potent nitric oxide synthase inhibitor, and given injections of 1.5 μg/kg PAF 15 min later. Animals treated with normal saline placebo, L-NAME alone, and PAF alone were also studied. Superior mesenteric artery blood flow and blood pressure were continuously recorded. At the end of 2 h or upon death of the animal, hematocrit was measured and intestinal samples were taken for histologic examination and determination of myeloperoxidase activity, a measure of intestinal neutrophil content. Compared with animals given PAF alone, animals pretreated with L-NAME followed by PAF developed significantly worse bowel injury (median injury scores: 2.5 versus 0.5, p = 0.005), hemoconcentration (Final hematocrit 65.2 ± 2.0% versus 53.9 ± 1.0%, p < 0.001), and intestinal myeloperoxidase activity (12.45 ± 1.94 U/g versus 6.51 ± 0.57 U/g, p < 0.01). The last two effects were further accentuated when 10 mg/kg L-NAME was given before PAF. Treatment with sodium nitroprusside, a nitric oxide donor, for 10 min before and after PAF administration reversed the effects of L-NAME. Animals pretreated with phenylephrine rather than L-NAME did not develop worse injury than animals treated with PAF alone despite comparable reductions in superior mesenteric blood flow before PAF treatment. Treatment with superoxide dismutase and catalase did not ameliorate the effects of L-NAME on PAF-induced injury, hemoconcentration, and neutrophil infiltration into the bowel. We conclude that endogenous nitric oxide defends against PAF-induced bowel injury by antagonizing certain immediate effects of PAF, particularly those leading to capillary leakage and neutrophil infiltration into the bowel. Our studies also suggest that the mechanism of action does not involve vasodilatation or scavenging of oxygen radicals.