Potential Cardio-Protective Agents: A Resveratrol Review (2000-2019)

With a 2030 projection of 23.6 million deaths per year, the prevalence and severity of cardiovascular disease are astoundingly high. Thus, there is a definitive need for the identification of novel compounds with the potential to prevent or treat the disease and associated states. Moreover, there is also an ever-increasing need for drug delivery systems (DDS) that cope with poor and ranging physiochemical properties of therapeutic compounds to achieve the clinical effect. The usage of resveratrol (RES) is a growing area of interest with innu-merate pieces of research, evidencing the drug’s efficacy. This drug is, however, marred; its notably poor phy-siochemical properties (namely poor water solubility) limit its use for oral drug delivery. RES analogues, how-ever, potentially possess superior physiochemical characteristics offering a remedy for the aforementioned drawback. However, particulate based DDS are equally able to offer property amelioration and targeting. This review offers an extensive examination into the role of RES as a potential cardioprotective agent. The prevalence and suitability of associated analogues and the role of nanotechnology in overcoming physicochemical boundaries, particularly through the development of nanoparticulate formulations, will be discussed in detail

Nanostructured lipid carriers deliver resveratrol, restoring attenuated dilation in small coronary arteries, via the AMPK pathway

Nanostructured lipid carriers (NLCs) are an emerging drug delivery platform for improved drug stability and the bioavailability of antihypertensive drugs and vasoprotective nutraceutical compounds, such as resveratrol (RV). The objective of this study was to ascertain NLCs’ potential to deliver RV and restore attenuated dilator function, using an ex vivo model of acute hyperten-sion. Trimyristin–triolein NLCs were synthesized and loaded with RV. The uptake of RV-NLCs by human coronary artery endothelial cells (HCAECs) maintained their viability and reduced both mitochondrial and cytosolic superoxide levels. Acute pressure elevation in isolated coronary arteries significantly attenuated endothelial-dependent dilator responses, which were reversed following incubation in RV-NLCs, superoxide dismutase or apocynin (p < 0.0001). RV-NLCs demonstrated a five-fold increase in potency in comparison to RV solution. At elevated pressure, in the presence of RV-NLCs, incubation with Nω-nitro-l-arginine (L-NNA) or indomethacin resulted in a significant reduction in the restored dilator component (p < 0.0001), whereas apamin and TRAM-34 had no overall effect. Incubation with the adenosine monophosphate-activated protein kinase (AMPK) inhibitor dorsomorphin significantly attenuated dilator responses (p < 0.001), whereas the SIRT-1 inhibitor EX-527 had no effect. RV-NLCs improved the impaired endothelial-dependent dilation of small coronary arteries, following acute pressure elevation, via NO and downstream COX elements, mediated by AMPK. We suggest that RV-NLCs are an effective delivery modality for improved potency and sustained drug release into the vasculature. Our findings have important implications for the future design and implementation of antihypertensive treatment strategies

Impact of phospholipids, surfactants and cholesterol selection on the performance of transfersomes vesicles using medical nebulizers for pulmonary drug delivery

The aim of this study is to formulate and optimize novel transfersome formulations for pulmonary drug delivery. Transfersome formulations (F1 – F18) were prepared by a thin-film method using three phospholipids (Soya phosphatidylcholine (SPC), Dimyristoly phosphatidylcholine (DMPC) and Hydrogenated soya phosphatidylcholine (HSPC)), in combination with three different surfactants (Tween 80, Span 80 and Span 20) with or without cholesterol, employing Beclomethasone dipropionate (BDP) as the model drug. Nano-transfersome formulations post-extrusion were delivered to a Two-stage Impinger (TSI) via three medical nebulizers (i.e. Air-jet, Ultrasonic and Vibrating mesh nebulizer). Based on the physicochemical properties, formulations F1 (SPC and Tween 80), F7 (DMPC and Tween 80) and F13 (HSPC and Tween 80) demonstrated significantly smaller VMD (162.34 ± 6.48, 198.66 ± 6.64, and 183.52 ± 7.34 nm), and significantly higher entrapment efficiency (97.56 ± 2.45, 95.67 ± 4.26 and 95.06 ± 3.38%). Based on nebulization performance, the Ultrasonic nebulizer exhibited the shortest nebulization time for formulations F1, F7 and F13 (i.e. 17.88 ± 2.45, 19.26 ± 2.04 and 19.59 ± 2.12 min), and higher output rate (212.04 ± 11.54, 194.61 ± 10.27 and 192.43 ± 9.84 mg/min), when compared to Air-jet and Vibrating mesh nebulizers. Irrespective of nebulizer type, significantly higher BDP deposition was observed in the lower stage of TSI for the F1 formulation (on average of 61%), whereas a higher amount of BDP was deposited in the upper stage of TSI using the F7 formulations (49%). Moreover, Formulation F1 in combination with Air-jet nebulizer demonstrated higher emitted dose (ED) and fine particle fraction (FPF) (82% and 83%), when compared to the counterpart formulations and nebulizer types investigated. This study has demonstrated that based on nebulizer performance, BDP deposition and formulation type; the F1 formulation in combination with an Air-jet nebulizer is most optimal for lower respiratory tract deposition, whereas the F7 formulation in combination with an Ultrasonic nebulizer is ideal for upper respiratory tract deposition

Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified γ-Secretase Complexes

BACKGROUND: Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1-42/Aβ1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1-42 production, decreasing Aβ1-40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1-40, Aβ1-42 and APP intracellular domain productions in vitro. CONCLUSION/SIGNIFICANCE: Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1-42/Aβ1-40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease

Selective neutralization of APP-C99 with monoclonal antibodies reduces the production of Alzheimer's Aβ peptides

The toxic amyloid-β (Aβ) peptides involved in Alzheimer's disease (AD) are produced after processing of the amyloid precursor protein-C-terminal fragment APP-C99 by γ-secretase. Thus, major therapeutic efforts have been focused on inhibiting the activity of this enzyme. However, preclinical and clinical trials testing γ-secretase inhibitors revealed adverse side effects most likely attributed to impaired processing of the Notch-1 receptor, a γ-secretase substrate critically involved in cell fate decisions. Here we report an innovative approach to selectively target the γ-secretase-mediated processing of APP-C99 with monoclonal antibodies neutralizing this substrate. Generated by immunizing mice with natively folded APP-C99, these antibodies bound N- or C-terminal accessible epitopes of this substrate, and decorated extracellular amyloid deposits in AD brain tissues. In cell-based assays, the same antibodies impaired APP-C99 processing by γ-secretase, and reduced Aβ production. Furthermore, they significantly decreased brain Aβ levels in the APPPS1 mouse model of AD after intracerebroventricular injection. Together, our findings support APP-C99 substrate-targeting antibodies as new immunotherapeutic and Notch-sparing agents to lower the levels of Aβ peptides implicated in AD

Enzymatic activity of highly purified γ-secretase complexes with FAD-linked or aspartate PS1 mutants.

<p>Equal amounts of the different purified γ-secretase preparations characterized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035133#pone-0035133-g004" target="_blank">Figure 4</a> were tested for activity on C100-Flag, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035133#pone-0035133-g003" target="_blank">Figure 3</a>. The resulting cleavage products were separated by SDS-PAGE and detected by immunostaining with an anti-Flag antibody (M2) for C100-Flag or AICD-Flag (A), and by sandwich ELISA for Aβ1–40 or Aβ1–42 (B). Note that the levels of Aβ produced from FAD-linked γ-secretase complexes were all in the non-linear range of the ELISA standards, close to the detection limit. Whenever possible, Aβ1–42/Aβ1–40 ratios were quantified and indicated on the top of the bars. Two independent purifications were performed on each clone and similar results were obtained. A representative dataset is shown.</p

Generation of stable cell lines overexpressing all human γ-secretase components with FAD-linked PS1 variants.

<p>MEF PS1/2^−/− were stably co-transduced with lentiviral vectors carrying genes encoding hNCT-V5, Flag-hPEN2, hAPH1aL-HA and clones were isolated by limiting dilution to generate a cell line, designated as γ- PS1/2, that overexpresses high amount of the three subunits. γ- PS1/2 MEFs were further transduced with hPS1 variants harbouring FAD-linked mutations or mutations in the catalytic aspartate residue(s), or PS1-WT, and cloned. Each clone, derived form the γ- PS1/2, was conveniently named according to the mutation present in PS1 preceded by the symbol γ and followed by the number of the clone (γ-MEF) in order to distinguish them from wild-type MEF (WT MEF) and MEF PS1/2^−/−. Two clones per γ-secretase variant were selected for characterization. (A–B) Whole cell protein extracts of the different cell lines were prepared in 1% NP40-HEPES buffer, separated by SDS-PAGE on 4–12% Bis-Tris or 12% Tris-Glycine gels and analysed by immunostaining to detect the γ-secretase core components NCT (NCT164), PS1 (NTF, MAB1563; CTF; MAB5232), APH1aL-HA (3F10), and Flag-PEN2 (M2) (A), and endogenous APP (A8717) (B). β-Actin was used as a loading control. Each lane represents one selected clone. <i>CTF</i>: C-terminal fragment, <i>FL</i>: full-length, <i>im.</i>: immature NCT; <i>m.</i>: mature NCT, <i>N</i>: N-glycosylated, <i>NTF</i>: N-terminal fragment, <i>O</i>: O-glycosylated.</p

High-grade purification of human γ-secretase complexes with FAD-linked PS1 mutants.

<p>(A) Schematic representation of the γ-secretase purification process. Briefly, Presenilin double-knockout MEFs were used to first generate cell lines that stably overexpress human γ-secretase complexes containing different PS1 variants. Next, these cell lines were used for a multi-step purification procedure as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035133#s4" target="_blank">material and methods</a>. (B) Blue-Native PAGE analysis of purified γ-secretase complexes made of different PS1 variants. Equal volumes of the different purified γ-secretase preparations were separated by native-PAGE on a 4–16% Bis-Tris gel, and stained with silver nitrate (top panel), or immunostained for NCT (NCT164, middle panel) or PS1-NTF (ab10281, bottom panel) as indicated. γ-Secretase complexes appeared on the gel as high molecular weight complexes (HMWCs) of ∼350 kDa. Note that the levels of HMWCs were similar for all clones. (C) Equal volumes of purified γ-secretase complexes with FAD-linked PS1 mutants were separated under denaturing conditions (SDS-PAGE) and immunostained with anti-NCT (NCT164), anti-PS1-NTF (MAB1563), anti-PS1-CTF (MAB5232), anti-HA (3F10), or anti-Flag (M2) antibodies. Two independent purifications were performed on each clone with similar results. A representative dataset is shown.</p