RESUMO
Atherosclerosis can be originated from the accumulation of modified cholesterol-rich lipoproteins in the arterial wall. The electronegative LDL, LDL(-), plays an important role in the pathogenesis of atherosclerosis once this cholesterol-rich lipoprotein can be internalized by macrophages, contributing to the formation of foam cells, and provoking an immune-inflammatory response. Herein, we engineered a nanoformulation containing highly pure surface-functionalized nanocapsules using a single-chain fragment variable (scFv) reactive to LDL(-) as a ligand and assessed whether it can affect the LDL(-) uptake by primary macrophages and the progression of atherosclerotic lesions in Ldlr -/- mice. The engineered and optimized scFv-anti-LDL(-)-MCMN-Zn nanoformulation is internalized by human and murine macrophages in vitro by different endocytosis mechanisms. Moreover, macrophages exhibited lower LDL(-) uptake and reduced mRNA and protein levels of IL1B and MCP1 induced by LDL(-) when treated with this new nanoformulation. In a mouse model of atherosclerosis employing Ldlr -/- mice, intravenous administration of scFv-anti-LDL(-)-MCMN-Zn nanoformulation inhibited atherosclerosis progression without affecting vascular permeability or inducing leukocytes-endothelium interactions. Together, these findings suggest that a scFv-anti-LDL(-)-MCMN-Zn nanoformulation holds promise to be used in future preventive and therapeutic strategies for atherosclerosis.
RESUMO
Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Polifenóis/farmacologia , Própole , Inibidores da Angiogênese/química , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Polifenóis/química , Própole/química , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
OBJECTIVE: Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. METHODS AND RESULTS: The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 µmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. CONCLUSION: Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.