Antithrombotic nanoparticles and methods for treating necrotizing enterocolitis

The present disclosure is directed to antithrombotic nanoparticles and methods of treating necrotizing enterocolitis

Cytokines and growth factors in the developing intestine and during necrotizing enterocolitis.

Cytokines and growth factors play diverse roles in the uninflamed fetal/neonatal intestinal mucosa and in the development of inflammatory bowel injury during necrotizing enterocolitis (NEC). During gestational development and the early neonatal period, the fetal/premature intestine is exposed to high levels of many inflammatory cytokines and growth factors, first via swallowed amniotic fluid in utero and then, after birth, in colostrum and mother\u27s milk. This article reviews the dual, seemingly counter-intuitive roles of cytokines, where these agents play a trophic role and promote maturation of the uninflamed mucosa, but can also cause inflammation and promote intestinal injury during NEC

All-Trans Retinoic Acid Induces TGF-β2 in Intestinal Epithelial Cells via RhoA- and p38α MAPK-Mediated Activation of the Transcription Factor ATF2.

We have shown previously that preterm infants are at risk of necrotizing enterocolitis (NEC), an inflammatory bowel necrosis typically seen in infants born prior to 32 weeks' gestation, because of the developmental deficiency of transforming growth factor (TGF)-β2 in the intestine. The present study was designed to investigate all-trans retinoic acid (atRA) as an inducer of TGF-β2 in intestinal epithelial cells (IECs) and to elucidate the involved signaling mechanisms.AtRA effects on intestinal epithelium were investigated using IEC6 cells. TGF-β2 expression was measured using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blots. Signaling pathways were investigated using Western blots, transiently-transfected/transduced cells, kinase arrays, chromatin immunoprecipitation, and selective small molecule inhibitors.AtRA-treatment of IEC6 cells selectively increased TGF-β2 mRNA and protein expression in a time- and dose-dependent fashion, and increased the activity of the TGF-β2 promoter. AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2. AtRA increased phospho-ATF2 binding to the TGF-β2 promoter and increased histone H2B acetylation in the TGF-β2 nucleosome, which is typically associated with transcriptional activation.AtRA induces TGF-β2 expression in IECs via RhoA- and p38α MAPK-mediated activation of the transcription factor ATF2. Further studies are needed to investigate the role of atRA as a protective/therapeutic agent in gut mucosal inflammation

Both ROCK1 and p38α MAPK are required for atRA-mediated phosphorylation of ATF2 in IECs.

<p><b><i>A</i>.</b> Western blots show the effect of the ROCK inhibitor Y-27632 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Β-actin was used as the loading control. <b><i>B</i>.</b> Effect of the p38 inhibitor SB203580 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Data represent 3 separate experiments. <b><i>C</i>.</b> Bar-diagrams (means ± SE) summarize ROCK activity in IEC6 cells treated with atRA (10 μM, top panel), with Y-27632 (10 μM) followed by atRA (10 μM; middle panel), and SB203580 (10 μM) followed by atRA (10 μM; bottom panel). Data represent 3 separate experiments; * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

AtRA-induced TGF-β₂ expression in IECs is mediated via RhoA GTPase and ROCK1.

<p><i>A</i>. Representative Western blots show increased expression of activated RhoA (RhoA-GTP) in IEC6 cells treated with atRA × 4h. Activated RhoA was pulled down from cell lysates using Rhotekin-agarose beads. Bar-diagram (means ± SE) summarizes densitometric data. <i>Inset</i>: Left panel: ATRA also increased the expression of total RhoA in IECs. Right panel: AtRA-treatment did not increase Rac1-GTP in IEC6 cells. <i>B</i>. Western blots show that atRA-induced TGF-β₂ expression in IEC6 cells was reproduced by over-expression of the constitutively-active GL4V mutant of RhoA. <i>C</i>. Cells expressing the TN19 dominant-negative RhoA mutant did not show atRA-induced TGF-β₂ expression. <i>D</i>. Western blots show cleaved ROCK1 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data. <i>Inset</i>: Fluorescence photomicrographs (magnification 630x) show nuclear localization of ROCK1 (green) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI (blue). <i>E</i>. Pharmacological inhibition of ROCK1 by Y-27632 blocked atRA-induced TGF-β₂ expression in IEC6 cells. Western blots show TGF-β₂ and cleaved ROCK1 expression. Bar-diagram (means ± SE) summarizes densitometric data, normalized against β-actin. Data represent 3 separate experiments; * <i>p</i><0.05 compared to cells cultured in media alone; # indicates <i>p</i><0.05 compared to atRA-treated cells.</p

AtRA modulates Smad expression in IECs.

<p><b><i>A</i>.</b> Western blots show the expression of Smad2, Smad3, Smad4, and Smad7 in atRA-treated IEC6 cells, shown as a function of time. <b><i>B</i>.</b> Bar-diagrams (means ± SE) summarize densitometric data of blots in panel A, normalized against β-actin. <b><i>C</i>.</b> Western blots show phospho-Smad2 expression as a function of time after atRA treatment. Data represent 3 separate experiments; * <i>p</i><0.05.</p

Transforming Growth Gactor-β\u3csub\u3e2\u3c/sub\u3e is Sequestered in Preterm Human Milk by Chondroitin Sulfate Proteoglycans

Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2 (rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2 (20–40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk

AtRA promotes the acetylation of histone H2B in the TGF-β₂ nucleosome.

<p><b><i>A</i>.</b> Western blots show the genome-wide acetylation status of histones H2A, H2B, H3, and H4. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. <b><i>B</i>.</b> AtRA treatment increases the acetylation of histone H2B (lys5) on the TGF-β₂ nucleosome. Bar diagram (means ± SE) shows data from ChIP assay, where the acetylated histones were pulled down and the presence of the TGF-β₂ promoter region in the complex confirmed by real-time PCR. Quantification of acetyl-H2B is shown as % input. <i>Inset</i>: Agarose gel showing enrichment of acetyl-H2B in the TGF-β₂ promoter. Data represent 3 separate experiments; * <i>p</i><0.05, *** <i>p</i><0.001.</p