px-478 and Burns

Ethanol remains a confounder in postburn pathology, which is associated with an impaired intestinal barrier. Previously, we demonstrated that ethanol and burn injury reduce intestinal oxygen delivery (hypoxia) and alters microRNA (miR) expression in small intestinal epithelial cells. Hypoxia has been shown to influence expression of miRs and miR biogenesis components. Therefore, we examined whether hypoxia influences expression of miR biogenesis components (drosha, dicer, and argonaute-2 [ago-2]) and miRs (-7a and -150) and whether these changes impacted other parameters following ethanol and burn injury. Mice were gavaged with ethanol (∼2.9 g/kg) 4 h before receiving a ∼12.5% total body surface full thickness burn. Mice were resuscitated at the time of injury with normal saline with or without 5 mg/kg PX-478, a hypoxia-inducible factor-1α inhibitor. One day following injury mice were euthanized, and the expression of miRs and their biogenesis components as well as bacterial growth, tight junction proteins, intestinal transit, and permeability were assessed. Ethanol combined with burn injury significantly reduced expression of drosha, ago-2, miRs (-7a and -150), occludin, zonula occludens-1, claudin-4, zonula occludens-1, mucins-2 and -4, and intestinal transit compared to shams. Furthermore, there was an increase in intestinal permeability, total bacteria, and Enterobacteriaceae populations following the combined injury compared to shams. PX-478 treatment improved expression of drosha, ago-2, miRs (-7a and -150), occludin, claudin-4, zonula occludens-1, and mucin-2. PX-478 treatment also improved intestinal transit and reduced dysbiosis and permeability. These data suggest that PX-478 improves miR biogenesis and miR expression, and restores barrier integrity while reducing bacterial dysbiosis following ethanol and burn injury.

Topics: Alcoholic Intoxication; Animals; Argonaute Proteins; Biomarkers; Burns; Disease Susceptibility; Enzyme Inhibitors; Ethanol; Hypoxia-Inducible Factor 1, alpha Subunit; Intestinal Mucosa; Male; Mice; MicroRNAs; Mustard Compounds; Phenylpropionates; Protective Agents; RNA, Messenger; RNA, Ribosomal, 16S

Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α(fl:fl)) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.

Topics: Activin Receptors, Type I; Adipose Tissue; Animals; Burns; Chondrogenesis; Disease Models, Animal; Gene Regulatory Networks; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Integrases; Luminescent Measurements; Mesenchymal Stem Cells; Mice, Knockout; Models, Biological; Mustard Compounds; Ossification, Heterotopic; Phenylpropionates; Receptor, Platelet-Derived Growth Factor alpha; RNA, Messenger; Signal Transduction; Sirolimus; SOX9 Transcription Factor; Tendons; Tenotomy; Up-Regulation; Wound Healing; Wounds and Injuries; X-Ray Microtomography