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

Non-polar secondary metabolites and essential oil of ex situ propagated and cultivated Sideritis syriaca L. subsp. syriaca (Lamiaceae) with consolidated identity (DNA Barcoding): towards a potential new industrial crop

Over the past decade, perennial plants of the genus Sideritis L. have attracted great scientific interest and they have become the subject of several studies, including the European Union's herbal monograph. The present study aimed to explore the identity of a biotype of S. syriaca subsp. syriaca documented with DNA barcoding, investigated its macro- and micro- propagation and to study the chemical profile of the dichloromethane extract, as well as of the Essential Oil (EO) of the ex situ propagated and cultivated plant material. An improved in vitro propagation protocol, as well as pioneer effective propagation with cuttings (both at commercially acceptable rates) are provided for a DNA-barcoded biotype of Sideritis syriaca L. subsp. syriaca (malotira, Cretan mountain tea) which is a single-island endemic plant of Crete (Greece), with declining wild populations due to over collection. From the aerial parts of ex situ propagated and cultivated material (harvested in July) two phytosterols (β-sitosterol and stigmasterol), two ent-kaurene diterpenes; siderol and eubotriol, as well as one methylated flavone (xanthomicrol) have been isolated. This is the first time that eubotriol has been isolated from this taxon. The structures of the isolated compounds have been established by spectroscopic means and the complete Nuclear Magnetic Resonance (NMR) assignments for eubotriol are presented. Moreover, the essential oil was obtained by hydrodistillation of the plant material during the flowering stage and was analyzed by Gas Chromatography-Mass Spectrometry (GC–MS). Forty-four compounds were identified. The monoterpene hydrocarbons constituted the major fraction of the essential oil (34.2%), while β-phellandrene (18.5%) and kaur-15-ene (or ent-kaur-15-ene; 17.3%) were the main compounds. In conclusion, the above results document the first necessary steps taken for the ex situ conservation and sustainable exploitation of a new industrial crop with promising potential. © 2020 Elsevier B.V

Changes in the cellular fatty acid profile drive the proteasomal degradation of α-synuclein and enhance neuronal survival

Parkinson's disease is biochemically characterized by the deposition of aberrant aggregated α-synuclein in the affected neurons. The aggregation properties of α-synuclein greatly depend on its affinity to bind cellular membranes via a dynamic interaction with specific lipid moieties. In particular, α-synuclein can interact with arachidonic acid (AA), a polyunsaturated fatty acid, in a manner that promotes the formation of α-helix enriched assemblies. In a cellular context, AA is released from membrane phospholipids by phospholipase A2 (PLA2). To investigate the impact of PLA2 activity on α-synuclein aggregation, we have applied selective PLA2 inhibitors to a SH-SY5Y cellular model where the expression of human wild-type α-synuclein is correlated with a gradual accumulation of soluble oligomers and subsequent cell death. We have found that pharmacological and genetic inhibition of GIVA cPLA2 resulted in a dramatic decrease of intracellular oligomeric and monomeric α-synuclein significantly promoting cell survival. Our data suggest that alterations in the levels of free fatty acids, and especially AA and adrenic acid, promote the formation of α-synuclein conformers which are more susceptible to proteasomal degradation. This mechanism is active only in living cells and is generic since it does not depend on the absolute quantity of α-synuclein, the presence of disease-linked point mutations, the expression system or the type of cells. Our findings indicate that the α-synuclein-fatty acid interaction can be a critical determinant of the conformation and fate of α-synuclein in the cell interior and, as such, cPLA2 inhibitors could serve to alleviate the intracellular, potentially pathological, α-synuclein burden. © 2020 Federation of American Societies for Experimental Biolog

Exploiting the role of hypoxia-inducible factor 1 and pseudohypoxia in the myelodysplastic syndrome pathophysiology

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoi-etic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS. © 2021 by the authors. Licensee MDPI, Basel, Switzerland