Development of an aptamer-conjugated fluorescent nanoprobe for MMP2

Matrix metalloproteinase 2 (MMP2) plays critical roles in various diseases, such as atherosclerosis and cancer, and has been suggested to contribute to the instability of atherosclerotic plaque. To visualize MMP2 in pathologic tissues, we developed an aptamer targeting MMP2 protein by performing eight rounds of modified DNA systematic evolution of ligands by exponential enrichment (SELEX). The aptamer showed high affinity for MMP2 (K_d = 5.59 nM), precipitated MMP2, and detected MMP2 protein in pathological tissues such as atherosclerotic plaque and gastric cancer tissues. Furthermore, a MMP2 aptamer-conjugated fluorescent nanoprobe successfully visualized atherosclerotic plaques in apolipoprotein E (ApoE) knockout mice. These results suggest that the devised MMP2 aptamer could be useful for the development of various diagnostic tools.     

Effect of Hibiscus anthocyanins-rich extract induces apoptosis of proliferating smooth muscle cell via activation of P38 MAPK and p53 pathway

Hibiscus sabdariffa L. (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in Sudan and in eastern Taiwan. It has been reported to contain a number of protocatechuic acid and anthocyanins. In vitro experimental studies have shown that anthocyanins administration of the extract produces anti-inflammation and chemoprevention effects. In spite of the wide use of Hibiscus sabdariffa L. in folk medicine for treating various diseases, our previous study indicated a potency of Hibiscus sabdariffa extract (HSE) in anti-atherosclerosis. The mechanisms of anthocyanins administration of the extract produce from Hibiscus sabdariffa L. to attenuate atherosclerosis were not clarified. In this study, we found that Hibiscus anthocyanins (HAs) could inhibit the serum-stimulated proliferation of smooth muscle cell (SMC) and result in cell apoptosis. The HAs inducing cell apoptosis was dose dependent. We further used SB203580 (p38 inhibitor) to block cellular apoptosis and evaluate its effect on the HAs-inducing SMC death via some apoptosis criteria including DNA fragmentation and flow cytometry. We suggested that the mechanisms of the inhibitory effect of HAs on atherosclerosis could be via inhibiting the proliferation of SMC. HAs induces apoptosis via (i) activating p38 MAP kinase that subsequently phosphorylates target protein c-Jun and transduces the signal to further activate the apoptotic protein cascades that contain Fas-mediated signaling (Fas/caspase-8 signaling module) and (ii) activating p53 and inducing bax expression. As an outcome of the events, cytochrome c releases from the mitochondria, leading to cell apoptosis. In these experiments, HAs showed strong potential to induce SMC cell apoptosis via p38 and p53 pathway. In consequence, the rate of atherosclerotic formation is slowed down, and the progress is suppressed.     

Effects of red grape skin and seed extract supplementation on atherosclerosis in Watanabe heritable hyperlipidemic rabbits

Epidemiological studies have suggested an association between consumption of red wine and other polyphenolic compounds and prevention of cardiovascular diseases. In the present study, Watanabe heritable hyperlipidemic (WHHL) rabbits were used to investigate the effects of polyphenols in a red grape skin and seed extract (GSE) on the development of atherosclerosis. WHHL rabbits received either semisynthetic diet (casein based) or semisynthetic diet added GSE over a period of 15 wk. Plasma lipids and aortic cholesterol accumulation were measured. Feeding semisynthetic diet was associated with increasing hypercholesterolemia, which was developing slower in GSE group compared to the controls as recorded by significantly lower plasma cholesterol in dosage week 7 (males: P < 0.05, females: P < 0.01) and 11 (males: P < 0.01). Aortic atherosclerosis evaluated as the cholesterol content in aortic tissue was comparable in the control and GSE-dosed females, but it was significantly reduced in the abdominal part of GSE-dosed male compared to the controls (P < 0.05). In conclusion, feeding GSE extract to WHHL rabbits had no significant effects in females but was associated with transient less hypercholesterolemic response to semisynthetic diet and, furthermore, retarded the development of aortic atherosclerosis in males as demonstrated by significantly lower cholesterol content in the abdominal part.     

Effects of trans‐10,cis‐12 and cis‐9,trans‐11 CLA on atherosclerosis in apoE/LDLR−/− mice

The objective of our studies was to verify the potential health‐related, anti‐atherogenic potency of CLA isomers, fed to apolipoprotein E and LDL receptor double knockout mice (apoE/LDLR^?/?), representing a reliable model of atherogenesis. Additionally, the effect of CLA isomers on liver steatosis was observed. In a “long experiment” (LONG), 2‐month‐old mice with no atherosclerosis were randomly assigned to three experimental groups and fed for the next 4 months. In a “short experiment” (SHORT), 4‐month‐old mice, with pre‐established atherosclerosis, were randomly assigned to three experimental groups and fed for the next 2 months. The experimental diets were: AIN‐93G (control), AIN‐93G + 0.5% trans‐10,cis‐12 CLA (t10,c12), and AIN‐93G + 0.5% cis9,trans‐11 CLA (c9,t11). In both experiments, c9,t11 CLA increased mice body weight. In mice fed t10,c12 CLA weight of liver was threefold (p<0.05) increased what was linked with hepatic steatosis observed in LONG and SHORT experiment. In LONG experiment, t10,c12 CLA significantly (p<0.05) increased plasma TAGs, whereas no such effect was observed in SHORT one. In mice receiving the CLA isomers the level of PPARα and SREBP‐1 mRNA in liver were significantly decreased. The expression of their target genes like ACO (PPARα) or FAS (SREBP‐1) were not changed. Only c9,t11 increased ACO level in LONG experiment. There were no isomer‐specific effects of CLA isomers on the area of atherosclerotic plaque. In conclusion, our results do not support the notion that CLA isomers supplementation to the diet has anti‐atherosclerotic effects. CLA isomers have no effect on atherosclerosis in apoE/LDLR^?/? mice. Practical applications: CLAs have been shown to occur naturally in food. In the last 10 years, attempts have been made to enrich animal‐derived foods in CLA isomers through animal nutrition strategies. Indeed, these attempts resulted in production of functional food such as CLA‐enriched milk (butter and cheese), ruminant and non‐ruminant meat, as well as eggs. In addition to natural foodstuff, dietary CLA supplements can also contribute to CLA intake in humans. Commercial CLA preparations, fed to laboratory animals, showed several health‐related properties, including anti‐adipogenic, anti‐carcinogenic, anti‐atherogenic, and anti‐inflammatory effects. The underlying mechanisms of action, however, are only poorly understood. The major objective of our studies was to verify the potential health‐related, namely anti‐atherogenic potency of CLA isomers, fed to apoE/LDLR^?/? mice, representing unique and reliable model of atherogenesis. Additionally, effect of CLA isomers on steatosis was observed.     

Detection of oxysterols in oxidatively modified low density lipoprotein by MALDI‐TOF MS

A simple method for the detection of oxysterols in oxidatively modified LDL (Ox‐LDL) has been developed using MALDI‐TOF MS. To identify the ion peaks of oxysterols, seven major oxysterols in Ox‐LDL (7α‐hydroxycholesterol, 7β‐hydroxycholesterol, 7‐ketocholesterol, 5α,6α‐epoxycholesterol, 5β,6β‐epoxycholesterol, 25‐hydroxychokesterol, (25R)‐26‐hydroxycholesterol), and cholesta‐3,5‐dien‐7‐one were analyzed by MALDI‐TOF MS. Among these oxysterols, 7‐ketocholesterol, a very abundant oxysterol in Ox‐LDL, was found to show a characteristic peak of [M + H]⁺ at m/z 401. Cholesta‐3,5‐dien‐7‐one, which is known as a degradation product of 7‐ketocholesterol upon saponification of Ox‐LDL, gave a major peak of [M + H]⁺ at m/z 383. In contrast, other oxysterols showed similar peak patterns at m/z 367 and 385. These results were applied to the analysis of Ox‐LDL by MALDI‐TOF MS after saponification and hexane‐extraction, detecting ion peaks at m/z 367, 383, 385, and 401. This MALDI‐TOF MS method has a potential as a simple tool to show the presence of oxysterols in Ox‐LDL without derivatization and chromatographic separation.     

The relation of lipid peroxidation processes with atherogenesis: a new theory on atherogenesis

The extremely high sensitivity of polyunsaturated fatty acids (PUFAs) to oxygen is apparently used by nature to induce stepwise appropriate cell responses. It is hypothesized that any alteration in the cell membrane structure induces influx of Ca2+ ions. Ca2+ ions are required to activate degrading enzymes, such as phospholipases and lipoxygenases (LOX) that transform PUFAs bound to membrane phospholipids to lipidhydroperoxides (LOOHs). Enzymatic reduction products of LOOHs seem to serve as ligands of proteins, which induce gene activation to initiate a physiological response. Increasing external impact on cells is connected with deactivation of LOX, liberation of the iron ion in its active center followed by cleavage of LOOH molecules to LO * radicals. LO * radicals induce a second set of responses leading to generation of unsaturated aldehydic phospholipids and unsaturated epoxyhydroxy acids that contribute to induction of apoptosis. Finally peroxyl radicals are generated by attack of LO * radicals on phospholipids. The latter attack nearly all types of cell constituents: Amino- and hydroxyl groups are oxidized to carbonyl functions, sugars and proteins are cleaved, molecules containing double bonds such as unsaturated fatty acids or cholesterol suffer epoxidation. LOOH molecules and iron ions at the cell wall of an injured cell are in tight contact with phospholipids of neighboring cells and transfer to these reactive radicals. Thus, the damaging processes proceed and cause finally necrosis except the chain reaction is stopped by scavengers, such as glutathione. Consequently, PUFAs incorporated into phospholipids of the cell wall are apparently equally important for the fate of a single organism as the DNA in the nucleus for conservation of the species. This review intends to demonstrate the connection of cell alteration reactions with induction of lipid peroxidation (LPO) processes and their relation to inflammatory diseases, especially atherosclerosis and a possible involvement of food. Previously it was deduced that food rich in cholesterol and saturated fatty acids is atherogenic, while food rich in n-3 PUFAs was recognized to be protective against vascular diseases. These deductions are in contradiction to the fact that saturated fatty acids withstand oxidation while n-3 PUFAs are subjected to LPO like all other PUFAs. Considering the influence of minor food constituents a new theory about atherogenesis and the influence of n-3 PUFAs is represented that might resolve the contradictory results of feeding experiments and chemical experiences. Cholesterol-PUFA esters are minor constituents of mammalian derived food, but main components of low density lipoprotein (LDL). The PUFA part of these esters occasionally suffers oxidation by heating or storage of mammalian derived food. There are indications that these oxidized cholesterol esters are directly incorporated into lipoproteins and transferred via the LDL into endothelial cells where they induce damage and start the sequence of events outlined above. The deduction that consumption of n-3 PUFAs protects against vascular diseases is based on the observation that people living on a fish diet have a low incidence to be affected by vascular diseases. Fish are rich in n-3 PUFAs; thus, it was deduced that the protective properties of a fish diet are due to n-3 PUFAs. Fish, fish oils, and vegetables contain besides n-3 PUFAs as minor constituents furan fatty acids (F-acids). These are radical scavengers and are incorporated after consumption of these nutrients into human phospholipids, leading to the assumption that not n-3 PUFAs, but F-acids are responsible for the beneficial efficiency of a fish diet.     

Tomato (Solanum lycopersicum L.) and type 2 diabetes

Since the late 1990s, various basic studies and clinical research have linked tomato to type 2 diabetes. This link, however, has yet to be understood and summarized. The present review systematically summarizes the effect of tomato on type 2 diabetes. The PubMed, Web of Science, and Embase databases were searched for reports published (in the English language) from December 1999 to August 2017 using the keywords “tomato” and “diabetes.” Relevant references from particular publications were also considered. To date, studies in humans have not shown a significant relationship between tomato and the risk of type 2 diabetes, whereas mainstream in vivo system studies have generally shown that tomato or its bioactive compounds may have an antihyperglycemic effect. Nevertheless, tomato (fresh or cooked) has been found to be favorable for diabetic conditions because it decreases diabetes-induced oxidative stress, inflammation, accelerated atherosclerosis, and tissue (i.e., retinal, renal, and musculoskeletal) damage. Further studies in humans are very important toward elucidating the hypoglycemic response of tomato or its derived compounds in diabetic conditions.     

Lipase C,Hepatic Type −250A/G (rs2070895) Variant Enhances Carotid Atherosclerosis in Normolipidemic and Asymptomatic Individuals from Brazil

The common genetic variant in the promoter region of the hepatic lipase gene [LIPC −250G/A(rs2070895)] has an ambiguous association with cardiovascular disease. In this context, our study was performed to identify the relationships between the rs2070895 with carotid atherosclerosis, plasma lipids, and parameters of reverse cholesterol transport. A total of 285 normolipidemic and asymptomatic participants from an initial sample of 598,288 individuals (inclusion criteria: LDL-C ≤130 mg/dL and triglycerides ≤150 mg/dL; age: 20–75 years, both genders; confirmation of clinical, anthropometric and laboratory data; attended all visits; DNA was achieved to perform genetic analysis) were enrolled and the rs2070895 variant was genotyped by TaqMan® OpenArray® Plataform. Carotid intima-media thickness and the screening of atherosclerotic plaques were determined by B-mode ultrasonography. The rs2070895 genotype frequencies were 0.44, 0.41, and 0.15 (GG, GA, and AA, respectively). Logistic regression analysis showed that the risk of having plaques was increased in participants carrying the AA or AG genotypes (OR = 3.90; 95% CI = 1.54–10.33), despite an increase in high-density lipoprotein cholesterol levels, HDL diameter and apolipoprotein A-I, as compared to the GG genotype. Hepatic lipase and endogenous lecithin cholesterol acyl transferase activities were reduced (38% and 19%, respectively) and lipoprotein lipase was increased by 30% (AA vs GG). Our results provide evidence that the AA or AG genotypes of the rs2070895 were associated with carotid atherosclerosis in apparently healthy participants, probably as a consequence of reduced reverse cholesterol transport and accumulation of HDL subfraction 2 rich in triglycerides and depleted in cholesteryl esters that could become dysfunctional.     

Network Medicine Approach in Prevention and Personalized Treatment of Dyslipidemias

Dyslipidemias can affect molecular networks underlying the metabolic homeostasis and vascular function leading to atherogenesis at early stages of development. Since disease-related proteins often interact with each other in functional modules, many advanced network-oriented algorithms were applied to patient-derived big data to identify the complex gene–environment interactions underlying the early pathophysiology of dyslipidemias and atherosclerosis. Both the proprotein convertase subtilisin/kexin type 7 (PCSK7) and collagen type 1 alpha 1 chain (COL1A1) genes arose from the application of TFfit and WGCNA algorithms, respectively, as potential useful therapeutic targets in prevention of dyslipidemias. Moreover, the Seed Connector algorithm (SCA) algorithm suggested a putative role of the neuropilin-1 (NRP1) protein as drug target, whereas a regression network analysis reported that niacin may provide benefits in mixed dyslipidemias. Dyslipidemias are highly heterogeneous at the clinical level; thus, it would be helpful to overcome traditional evidence-based paradigm toward a personalized risk assessment and therapy. Network Medicine uses omics data, artificial intelligence (AI), imaging tools, and clinical information to design personalized therapy of dyslipidemias and atherosclerosis. Recently, a novel non-invasive AI-derived biomarker, named Fat Attenuation Index (FAI™) has been established to early detect clinical signs of atherosclerosis. Moreover, an integrated AI-radiomics approach can detect fibrosis and microvascular remodeling improving the customized risk assessment. Here, we offer a network-based roadmap ranging from novel molecular pathways to digital therapeutics which can improve personalized therapy of dyslipidemias.