The term angiogenesis refers to sprouting of new blood vessels from pre-existing ones. The angiogenic process involves cell migration and tubulogenesis requiring interaction between endothelial cells and the extracellular matrix. Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase found embedded in the basement membranes. As it promotes the stabilization of extracellular matrix, we investigated its possible role in angiogenesis both in vitro and in vivo. We analysed the effects of peroxidasin 1 gene silencing and supplementation by recombinant hsPxd01 in TeloHAEC endothelial cells on cell migration, tubulogenesis in matrigel, and intracellular signal transduction as assessed by kinase phosphorylation and expression of pro-angiogenic genes as measured by qRT–PCR. We further evaluated the angiogenic potential of recombinant peroxidasin in a chicken chorioallantoic membrane model. RNA silencing of endogenous hsPxd01 significantly reduced tube formation and cell migration, whereas supplementation by the recombinant peroxidase promoted tube formation in vitro and stimulated vascularization in vivo through its catalytic activity. Moreover, recombinant hsPxd01 promoted phosphorylation of Extracellular signal-Regulated Kinases (ERK1/2), Protein kinase B (Akt), and Focal Adhesion Kinase (FAK), and induced the expression of pro-angiogenic downstream genes: Platelet Derived Growth Factor Subunit B (PDGFB), endothelial-derived Heparin Binding EGF-like growth factor (HB-EGF), CXCL-1, Hairy-Related Transcription Factor 1 (HEY-1), DNA-binding protein inhibitor (ID-2), Snail Family Zinc Finger 1 (SNAI-1), as well as endogenous hsPxd01. However, peroxidasin silencing significantly reduced Akt and FAK phosphorylation but induced ERK1/2 activation after supplementation by recombinant hsPxd01. While hsPxd01 silencing significantly reduced expression of HEY-1, ID-2, and PDGFB, it did not affect expression of SNAI-1, HB-EGF, and CXCL-1 after supplementation by recombinant hsPxd01. Our findings suggest a role of enzymatically active peroxidasin 1 as a pro-angiogenic peroxidase and a modulator of ERK1/2, Akt and FAK signalling.

Background: Propolis is a resinous substance produced by bees and known to possess antioxidant, antimicrobial, antiproliferative and anti-inflammatory activities. Objective: This study is aimed at evaluating the in vivo and in vitro anti-inflammatory potential of the Crude Ethanolic Extract (CE) of Lebanese propolis and its Ethyl Acetate Fraction (EAF). Method: Chemical content of propolis was characterized using high-performance liquid chromatography and LC-MS/MS. COX-2 and iNOS protein expression, nitric oxide (NO) and prostaglandin (PGE2) release in LPS-activated RAW monocytes were achieved respectively by western blot and spectrophotometry. Antioxidant activity was evaluated by DPPH free radical scavenging assay. Measurement of paw thickness in carrageenan-induced paw edema in mice and pathologic assessment of inflammation in paw sections were used to judge the anti-inflammatory properties of propolis. Results: Pathology analysis revealed in the treated group significant reduction of immune cell infiltration and edema. Both extract and ethyl acetate fraction showed significant anti-inflammatory and antioxidant effects in LPS-treated RAW cells characterized by the inhibition of COX-2 and iNOS protein expression, as well as PGE2 and NO release. Chemical analysis of the crude extract and its ethyl acetate fraction identified 28 different compounds of which two phenolic acids and nine other flavonoids were also quantified. Ferulic acid, caffeic acid, chrysin, galangin, quercetin, and pinocembrin were among the most representative compounds. Conclusion: Lebanese propolis is rich in a various amount of flavonoids which showed promising antiinflammatory and antioxidant properties. Additionally, chemical analysis showed unique chemical compositions with the potential of identifying ingredients with interesting anti-inflammatory activities.