px-478 and Disease-Models--Animal

Hypoxia inducible factor 1α (HIF1α) plays a critical role in atherosclerosis as demonstrated in endothelial-targeted HIF1α -deficient mice. However, it has not been shown if specific pharmacological inhibitors of HIF1α can be used as potential drugs for atherosclerosis. PX-478 is a selective inhibitor of HIF1α, which was used to reduce cancer and obesity in animal models. Here, we tested whether PX-478 can be used to inhibit atherosclerosis.. PX-478 treatment reduced atherosclerotic plaque burden in the aortic trees in both mouse models, while plaque burden in the aortic sinus was reduced in the AAV-PCSK9 mouse model, but not in the ApoE. These results suggest that PX-478 is a potential anti-atherogenic drug, which targets vascular endothelium and hepatic cholesterol pathways.

Topics: Animals; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Disease Models, Animal; Endothelial Cells; Hypoxia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mustard Compounds; Phenylpropionates; Plaque, Atherosclerotic; Proprotein Convertase 9

Phosphatase and tensin homolog (PTEN) deleted on human chromosome 10 is a tumor suppressor with bispecific phosphatase activity, which is often involved in the study of energy metabolism and tumorigenesis. PTEN is recently reported to participate in the process of acute injury. However, the mechanism of PTEN in Ischemia-Reperfusion Injury (IRI) has not yet been clearly elucidated. In this study, mice with bilateral renal artery ischemia-reperfusion and HK-2 cells with hypoxia/reoxygenation (H/R) were used as acute kidney injury models. We demonstrated that PTEN was downregulated in IRI-induced kidney as well as in H/R-induced HK-2 cells. By silencing and overexpressing PTEN with si-PTEN RNA and PHBLV-CMV-PTEN-flag lentivirus before H/R, we found that PTEN protected HK-2 cells against H/R-induced injury reflected by the change in cell activity and the release of LDH. Furthermore, we inhibited HIF1-α with PX-478 and inactivated mTOR with Rapamycin before the silence of PTEN in H/R model. Our data indicated that the renoprotective effect of PTEN worked via PI3K/Akt/mTOR pathway and PI3K/Akt/HIF1-α pathway, hence alleviating apoptosis and improving autophagy respectively. Our findings provide valuable insights into the molecular mechanism underlying renoprotection of PTEN on autophagy and apoptosis induced by renal IRI, which offers a novel therapeutic target for the treatment of AKI.

Topics: Acute Kidney Injury; Animals; Apoptosis; Autophagy; bcl-2-Associated X Protein; Cell Line; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Male; Mice; Mice, Inbred C57BL; Mustard Compounds; Phenylpropionates; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Reperfusion Injury; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Hypoxia-inducible factor-1α (Hif1α) is activated in hypoxia and is closely related to oxidative stress, immunity and cell metabolism. Recently, it is reported that Hif1α is involved in atherosclerosis, ischemia-reperfusion (I/R) injury, alcoholic liver disease and pancreatic tumors. In this study, we found that Hif1 signal pathway is significantly changed in pancreas of acute pancreatitis (AP) mice. Meanwhile, we verified that the high expression of Hif1α injured pancreatic tissues of cerulean-induced AP mice, which prompting that Hif1α participated in the progress of histopathology on AP. We applied a Hif1α inhibitor PX478 and observed that it could alleviate histological injury of pancreas as well as the levels of serum amylase, lipase and proinflammatory cytokine in the murine model of AP induced by caerulein. In addition, PX478 could reduce the formation of necrosome (RIP3 and p-MLKL) and the generation of reactive oxygen species (ROS) in AP mice. Correspondingly, we further confirmed the effectiveness of PX478 in vitro and found that inhibiting Hif1α could mitigated the necrosis of pancreatic acinar cells via reducing the RIP3 and p-MLKL expression and the ROS production. In conclusion, inhibiting Hif1α could protect against acinar cells necrosis in AP, which may provide a new target for the prevention and treatment of AP clinically.

Topics: Acinar Cells; Animals; Disease Models, Animal; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred ICR; Mustard Compounds; Necrosis; Pancreatitis; Phenylpropionates

Retinopathy of prematurity (ROP) is an important risk factor for blindness in children due to neovascularization (NV). Hypoxia stimulates the formation of NV, as retinal hypoxia affects the stability and function of hypoxia-inducible factor (HIF) transcription factors. The purpose of this study is to study the mechanism of ROP and provide theoretical basis for clinical treatment of ROP.. In the present study, we used a mouse model of oxygen-induced retinopathy (OIR) to demonstrate the effects of the HIF-1α inhibitor PX-478 on OIR, and to determine its mechanism of action, to provide a theoretical basis for the clinical treatment of ROP.. The OIR mouse model was induced by exposing neonatal mouse pups and their mothers to 75 ± 5% oxygen from postnatal day 7 (P7) to P12, before being returned to room air from P12 to P17. Flat mount analyses were performed at P12 and P17. Hif1a, Hif2a, Hif3a, and Vegfa mRNA were detected by reverse transcription-polymerase chain reaction in OIR mice at P12 and P17. Hif1a and Vegfa mRNA were detected in OIR mice at P12 and P17 treatment with PX-478. Western blot analyses were used to assess the levels of HIF-1α, VEGF-A, and EPO before and after treatment with PX-478 at P12 and P17.. Hif1a mRNA was increased in OIR mice at P12 and P17, while Vegfa mRNA was increased at P12 and P17. HIF-1α, VEGF-A, and EPO protein levels were increased in OIR mice at P12 and P17, as compared to control mice at the same age (all p < 0.05). Inhibition of HIF-1α by injection of PX-478 in OIR mice (P9-P16) caused a decrease in the retinal avascular area at P12 and P17 (both p < 0.05), NV areas at P17 (p < 0.05), Vegfa mRNA decreased at P12 and P17, as compared to control mice (p < 0.05), and VEGF-A and EPO protein levels at P12 and P17, as compared to control mice. Our study found that there were PX-478 both retina and vitreous body of OIR. Inhibition of HIF-1α by injection of PX-478 in OIR mice caused a decrease in the retinal avascular area at P12 and P17, NV areas decreased at P17, VEGF-A and EPO protein levels at P12 and P17. Endothelial cell migration assays and cell tube formation indication PX-478 attenuate cell migration and significantly weakened the cell cavity formation under the condition of hypoxia.. HIF-1α plays a main role in OIR and can be considered a therapeutic target in OIR by suppressing downstream angiogenic factors, PX-478 decreasing the retinal avascular area and NV.

Topics: Animals; Animals, Newborn; Blotting, Western; Disease Models, Animal; Female; Gene Expression Regulation, Developmental; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred C57BL; Mustard Compounds; Oxygen; Phenylpropionates; Retina; Retinopathy of Prematurity; RNA

Hepatic stellate cells (HSCs) are liver-specific pericytes regulating angiogenesis during liver fibrosis. We aimed to elucidate the mechanisms by which hedgehog signalling regulated HSC angiogenic properties and to validate the therapeutic implications.. Rats and mice were treated with carbon tetrachloride for in vivo evaluation of hepatic angiogenesis and fibrotic injury. Diversified molecular approaches including real-time PCR, Western blot, luciferase reporter assay, chromatin immunoprecipitation, electrophoretic mobility shift assay and co-immunoprecipitation were used to investigate the underlying mechanisms in vitro.. Angiogenesis was concomitant with up-regulation of Smoothened (SMO) and hypoxia inducible factor-1α (HIF-1α) in rat fibrotic liver. The SMO inhibitor cyclopamine and Gli1 inhibitor GANT-58 reduced expression of VEGF and angiopoietin 1 in HSCs and suppressed HSC tubulogenesis capacity. HIF-1α inhibitor PX-478 suppressed HSC angiogenic behaviour, and inhibition of hedgehog decreased HIF-1α expression. Furthermore, heat shock protein 90 (HSP90) was characterized as a direct target gene of canonical hedgehog signalling in HSCs. HSP90 inhibitor 17-AAG reduced HSP90 binding to HIF-1α, down-regulated HIF-1α protein abundance and decreased HIF-1α binding to DNA. 17-AAG also abolished 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) (a SMO agonist)-enhanced HSC angiogenic properties. Finally, the natural compound ligustrazine was found to inhibit canonical hedgehog signalling leading to suppressed angiogenic properties of HSCs in vitro and ameliorated liver fibrosis and sinusoidal angiogenesis in mice.. We have provided evidence that the canonical hedgehog pathway controlled HSC-mediated liver angiogenesis. Selective inhibition of HSC hedgehog signalling could be a promising therapeutic approach for hepatic fibrosis.

Topics: Animals; Benzoquinones; Carbon Tetrachloride; Disease Models, Animal; Hedgehog Proteins; Hepatic Stellate Cells; HSP90 Heat-Shock Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Lactams, Macrocyclic; Liver Cirrhosis; Male; Mice; Mice, Inbred ICR; Mustard Compounds; Neovascularization, Pathologic; Phenylpropionates; Pyrazines; Rats; Rats, Sprague-Dawley; Signal Transduction; Smoothened Receptor

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

During a course of colitis, production of the gaseous mediator hydrogen sulfide (H2S) is markedly up-regulated at sites of mucosal damage and contributes significantly to healing and resolution of inflammation. The signaling mechanisms through which H2S promotes resolution of colitis are unknown. We hypothesized that the beneficial effects of H2S in experimental colitis are mediated via stabilization of hypoxia-inducible factor (HIF)-1α. The hapten dinitrobenzene sulfonic acid was used to induce colitis in rats and mice. This resulted in an elevated expression of the H2S-producing enzyme, cystathionine γ-lyase (CSE), and HIF-1α at sites of mucosal ulceration, and the expression of these 2 enzymes followed a similar pattern throughout the course of colitis. This represented a functionally important relationship because the loss of CSE-derived H2S production led to decreased HIF-1α stabilization and exacerbation of colitis. Furthermore, application of an H2S-releasing molecule, diallyl disulfide (DADS), stabilized colonic HIF-1α expression, up-regulated hypoxia-responsive genes, and reduced the severity of disease during peak inflammation. Importantly, the ability of DADS to promote the resolution of colitis was abolished when coadministered with an inhibitor of HIF-1α in vivo (PX-478). DADS was also able to maintain HIF-1α expression at a later point in colitis, when HIF-1α levels would have normally returned to control levels, and to enhance resolution. Finally, we found that HIF-1α stabilization inhibited colonic H2S production and may represent a negative feedback mechanism to prevent prolonged HIF-1α stabilization. Our findings demonstrate an important link between H2S and HIF-1α in the resolution of inflammation and injury during colitis and provide mechanistic insights into the therapeutic value of H2S donors.

Topics: Allyl Compounds; Animals; Benzenesulfonates; Colitis; Cystathionine gamma-Lyase; Disease Models, Animal; Disulfides; Gene Expression; HT29 Cells; Humans; Hydrogen Sulfide; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred C57BL; Mustard Compounds; Phenylpropionates; Protein Stability; Rats; Rats, Wistar; Wound Healing