RESUMEN

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency of neonates. Epithelial tight junction (TJ) proteins, such as claudins, are essential for regulation and function of the intestinal barrier. Rho kinase (ROCK) affects cellular permeability and TJ regulation. We hypothesized that TJ protein changes would correlate with increased permeability in experimental NEC, and ROCK inhibitors would be protective against NEC by regulation of key claudin proteins. We tested this hypothesis using an in vivo rat pup model, an in vitro model of experimental NEC, and human intestinal samples from patients with and without NEC. Experimental NEC was induced in rats via hypoxia and bacteria-containing formula, and in Caco-2 cells by media inoculated with LPS. The expression of claudins was measured by gene and protein analysis. Experimental NEC in rat pups and Caco-2 cells had increased permeability compared to controls. Gene and protein expression of claudin 2 was increased in experimental NEC. Sub-cellular fractionation localized increased claudin 2 protein to the cytoskeleton. ROCK inhibition was associated with normalization of these alterations and decreased severity of experimental NEC. Co-immunoprecipitation of caveolin-1 with claudin 2 suggests that caveolin-1 may act as a shuttle for the internalization of claudin 2 seen in experimental NEC. In conclusion, NEC is associated with intestinal permeability and increased expression of claudin 2, increased binding of caveolin-1 and claudin 2, and increased trafficking of claudin 2 to the cytoskeleton.

Asunto(s)

RESUMEN

Necrotizing enterocolitis (NEC) is a deadly disease that occurs in 5-10% of neonates. Although NEC has been extensively studied, no single therapeutic target has been identified. Rho kinase (ROCK) is a serine/threonine kinase that affects multiple cellular processes, including tight junction (TJ) function, cellular permeability, and apoptosis. We hypothesized that ROCK inhibition would decrease cellular permeability, stabilize TJ proteins (occludin), and decrease the severity of NEC. To test this hypothesis, human colon epithelial cells (Caco-2) and human endothelial cells were studied. Cells were treated with lipopolysaccharide to simulate an in vitro model of NEC. The effect of ROCK inhibition was measured by transepithelial membrane resistance (TEER) and cellular permeability to FITC-dextran. The effects of ROCK inhibition in vivo were analyzed in the rat pup model of NEC. NEC was induced by feeding formula supplemented with Cronobacter sakazakii with or without gavaged ROCK inhibitor. Rat intestines were scored based on histological degree of injury. RNA and protein assays for occludin protein were performed for all models of NEC. Treatment with ROCK inhibitor significantly decreased cellular permeability in Caco-2 cells and increased TEER. Intestinal injury scoring revealed decreased scores in ROCK inhibitor-treated pups compared with NEC only. Both cell and rat pup models demonstrated an upregulation of occludin expression in the ROCK inhibitor-treated groups. Therefore, we conclude that ROCK inhibition protects against experimental NEC by strengthening barrier function via upregulation of occludin. These data suggest that ROCK may be a potential therapeutic target for patients with NEC. NEW & NOTEWORTHY These studies are the first to demonstrate an upregulation of occludin tight junction protein in response to Rho kinase (ROCK) inhibition. Furthermore, we have demonstrated that ROCK inhibition in experimental models of necrotizing enterocolitis (NEC) is protective against NEC in both in vitro and in vivo models of disease.

Asunto(s)

RESUMEN

Necrotizing enterocolitis (NEC) is a serious intestinal disease that occurs in newborn infants. It is associated with major morbidity and affects 5% of all infants admitted to neonatal intensive care units. Probiotics have variable efficacy in preventing necrotizing enterocolitis. Tight junctions (TJ) are protein complexes that maintain epithelial barrier integrity. We hypothesized that the probiotics Lactobacillus rhamnosus and Lactobacillus plantarum strengthen intestinal barrier function, promote TJ integrity, and protect against experimental NEC. Both an in vitro and an in vivo experimental model of NEC were studied. Cultured human intestinal Caco-2 cells were pretreated with L. rhamnosus and L. plantarum probiotics. TJ were then disrupted by EGTA calcium switch or LPS to mimic NEC in vitro. Trans-epithelial resistance (TER) and flux of fluorescein isothiocynate dextran was measured. TJ structure was evaluated by ZO-1 immunofluorescence. In vivo effects of ingested probiotics on intestinal injury and ZO-1 expression were assessed in a rat model of NEC infected with Cronobacter sakazakii (CS). Caco-2 cells treated with individual probiotics demonstrated higher TER and lower permeability compared to untreated cells (p<0.0001). ZO-1 immunofluorescence confirmed TJ stability in treated cells. Rat pups fed probiotics alone had more intestinal injury compared with controls (p=0.0106). Probiotics were protective against injury when given in combination with CS, with no difference in intestinal injury compared to controls (p=0.21). Increased permeability was observed in the probiotic and CS groups (p=0.03, p=0.05), but not in the probiotic plus CS group (p=0.79). Lactobacillus sp. strengthened intestinal barrier function and preserved TJ integrity in an in vitro experimental model of NEC. In vivo, probiotic bacteria were not beneficial when given alone, but were protective in the presence of CS in a rat model of NEC.

RESUMEN

Necrotizing enterocolitis (NEC) is a devastating intestinal disease that has been associated with Cronobacter sakazakii and typically affects premature infants. Although NEC has been actively investigated, little is known about the mechanisms underlying the pathophysiology of epithelial injury and intestinal barrier damage. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are important mediators and regulators of apoptosis. To test the hypothesis that C. sakazakii increases cAMP and PKA activation in experimental NEC resulting in increased epithelial apoptosis, we investigated the effects of C. sakazakii on cAMP and PKA in vitro and in vivo. Specifically, rat intestinal epithelial cells and a human intestinal epithelial cell line were infected with C. sakazakii, and cAMP levels and phosphorylation of PKA were measured. An increase in cAMP was demonstrated after infection, as well as an increase in phosphorylated PKA. Similarly, increased intestinal cAMP and PKA phosphorylation were demonstrated in a rat pup model of NEC. These increases were correlated with increased intestinal epithelial apoptosis. The additional of a PKA inhibitor (KT5720) significantly ameliorated these effects and decreased the severity of experimental NEC. Findings were compared with results from human tissue samples. Collectively, these observations indicate that cAMP and PKA phosphorylation are associated with increased apoptosis in NEC and that inhibition of PKA activation protects against apoptosis and experimental NEC.

Asunto(s)

RESUMEN

Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of gamma-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane.

Asunto(s)