Journal: Bioactive Materials

Article Title: ETV2 regulating PHD2-HIF-1α axis controls metabolism reprogramming promotes vascularized bone regeneration

doi: 10.1016/j.bioactmat.2024.02.014

Figure Lengend Snippet: Reduction of α-KG promotes angiogenesis (A) Representative images of immunofluorescence co-staining CD31 (red) and CD90 (green) in a mouse tooth extraction model. The white arrows indicate the close proximity of CD31 + cells and CD90 + cells. Scale bar: 100 μm (B) α-KG content in the culture supernatant of an equal number of NC-DPSCs and ETV2-DPSCs (C) Tube formation of HUVECs treated with an ETV2-conditioned medium, with or without the incorporation of α-KG or IOX4 (D) The wound healing assay conducted on HUVECs treated with ETV2-conditioned medium, both with and without α-KG or IOX4 incorporation (E) The CCK-8 assay of HUVECs treated by ETV2 conditioned medium with or without α-KG or IOX4 (F) The protein expression of PHD2, HIF-1α, and VE cadherin in HUVECs treated by ETV2 conditioned medium with or without α-KG or IOX4 (G, H) The statistical analysis of node numbers and relative migration rates in C and D (I) Macroscopic images and size analysis of resected Matrigel plug (J) Representative images and neovascularization region statistics of immunofluorescence labeling CD31 in Matrigel plug. The areas exhibiting positive staining are denoted by the white arrows. Scale bar: 100 μm (K) Schematic illustration of mechanism of vascularization (NC, negative control; OE, overexpression. Data are presented as the mean of >3 independent experiments ±SD. *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: Diverse cell supernatants, 100 μM/ml α-KG (MCE), and 10 nM/ml IOX4 (a competitive inhibitor of α-KG, MCE) were incorporated to diverse experimental groups.

Techniques: Immunofluorescence, Staining, Extraction, Wound Healing Assay, CCK-8 Assay, Expressing, Migration, Labeling, Negative Control, Over Expression

Journal: The Journal of Immunology Author Choice

Article Title: Nicotinamide Antagonizes Lipopolysaccharide-Induced Hypoxic Cell Signals in Human Macrophages

doi: 10.4049/jimmunol.2200552

Figure Lengend Snippet: NAM and LPS crosstalk. HIF-1α regulation: The stability of HIF-1α is regulated by cell signals that affect HIF-1α transcription, translation, and degradation. In the absence of hypoxic responses, pyruvate generated during glycolysis enters the mitochondria and fuels the citric acid cycle. Iron, oxygen, and α-ketoglutarate are metabolized in the mitochondria and are essential for the function of PHD2, the primary prolyl hydroxylase for HIF-1α. Hydroxylation of HIF-1α targets the protein to the VHL E3 ligase that functions with chaperone proteins to add ubiquitin to HIF-1α for targeted degradation by the 26S proteasome. Drugs that block the PHD2 α-ketoglutarate binding site (IOX4), sequester iron (deferoxamine mesylate [DFOM]), or block a catalytic site in the proteasome (MG-132) prevent HIF-1α degradation and promote HIF-1α stabilization. The LPS response: LPS induced HIF-1α transcription, which may occur indirectly through NF-κB and the acetylation of the p65 subunit. LPS induces the phosphorylation of AKT, which can induce downstream signaling that activates the NF-κB dimer unit (p50/p65). AKT also phosphorylates acetylated FOXO1 at Ser256, which promotes nuclear exclusion and degradation and blocks FOXO1 activity. LPS both promotes (SIRT1) and inhibits (HDAC4) the activity of deacetylases. LPS activates glycolytic genes and increases glycolysis, possibly through HIF-1α protein stability. LPS-induced hypoxic metabolism is reflected in the media by increased production of protons (H+) and increased cellular NADH but lower levels of cellular NAD. LPS stabilizes HIF-1α through increased PSMF1 production that reduces proteasome formation and NADPH oxidase activity that utilize oxygen to form free radicals. The NAM effect: NAM increases cellular NAD levels, reduces glycolysis (H+), and increases PHD2 activity/hydroxylation, which promotes HIF-1α ubiquitination through the VHL E3 ligase. NAM enhances formation of the proteasome (e.g., PSMF1 inhibition) for targeted HIF-1α degradation and antagonizes free radical production through NOX2. NAM increases p65 ubiquitination, inhibits LPS-induced phosphorylation of AKT and FOXO1, and reduces the acetylation of p65, possibly through increased NAD/SIRT1. Increased HDAC4 promotes the stability of the NF-κB inhibitor, IκBα, to further prevent NF-κB activation.

Article Snippet: Cells were resuspended (2 × 10 6 /ml) in IMDM (Life Technologies) supplemented with 10% FBS and 30 ng/ml M-CSF (PHC9504, Thermo Scientific) for 6 d. HMDMs were lifted and plated at 5 × 10 5 /ml in 10% FBS/IMDM overnight prior to additional cell stimulation with NAM (N0636, Sigma-Aldrich, suspended in media), MG-132 (M7449, Sigma-Aldrich, suspended in DMSO), IOX4 (S6684, Selleck Chemicals, suspended in DMSO), IDO1 inhibitor (S8557, Selleck Chemicals, suspended in DMSO), deferoxamine mesylate (DFOM; sc-203331, Santa Cruz, suspended in water), 1-methyl- d -tryptophan (1-MT; 452483, Sigma-Aldrich, 1 mM intermittently vortexed and incubated in warm media [37°C] until soluble), or LPS (433, List Biologicals, suspended in media) as detailed in representative figures.

Techniques: Generated, Ubiquitin Proteomics, Blocking Assay, Binding Assay, Phospho-proteomics, Activity Assay, Inhibition, Activation Assay