A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Transfection Mechanisms of Polyplexes, Lipoplexes, and Stealth Liposomes in α₅β₁ Integrin Bearing DLD‑1 Colorectal Cancer Cells

Receptor targeted, PEGylated transfection agents can improve stability and delivery specificity of current cationic lipid and polymer based nonviral gene delivery vehicles, but their mode of transfection is poorly understood. We therefore investigated the transfection mechanisms of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) lipoplexes, branched polyethylenimine (bPEI) polyplexes, and bPEI encapsulated in either PEGylated (stealth) nontargeted liposomes or PR_b peptide (targeted to α₅β₁ integrin) functionalized stealth liposomes in DLD-1 colorectal cancer cells in vitro with gene expression assays, flow cytometry and confocal microscopy. DOTAP/DOPE and PR_b functionalized stealth liposomes mediated higher gene expression compared to nontargeted stealth liposomes and bPEI. However DOTAP/DOPE was internalized slowly leading to lower levels of DNA uptake. In contrast, despite high internalization of bPEI polyplexes, gene expression levels were low as DNA was unable to escape from the endosomes. Nontargeted stealth liposomes also mediated low gene expression due to low amounts of DNA internalized and slow internalization kinetics. PR_b functionalized stealth liposomes struck an optimal balance among these transfection agents with efficient transfection arising from fast integrin mediated internalization kinetics, high amounts of DNA uptake, and endosomal escape. We found α₅β₁ integrin to be a valuable target for gene delivery and that the caveolar endocytic pathway may offer an advantage to receptor targeted PEGylated transfection agents in DLD-1 cells