Variability in the Clinical Effects of the Omega-3 Polyunsaturated Fatty Acids DHA and EPA in Cardiovascular Disease-Possible Causes and Future Considerations.

Cardiovascular disease (CVD) that includes myocardial infarction and stroke, is the leading cause of mortality worldwide. Atherosclerosis, the primary underlying cause of CVD, can be controlled by pharmacological and dietary interventions, including n-3 polyunsaturated fatty acid (PUFA) supplementation. n-3 PUFA supplementation, primarily consisting of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has shown promise in reducing atherosclerosis by modulating risk factors, including triglyceride levels and vascular inflammation. n-3 PUFAs act by replacing pro-inflammatory fatty acid types in cell membranes and plasma lipids, by regulating transcription factor activity, and by inducing epigenetic changes. EPA and DHA regulate cellular function through shared and differential molecular mechanisms. Large clinical studies on n-3 PUFAs have reported conflicting findings, causing confusion among the public and health professionals. In this review, we discuss important factors leading to these inconsistencies, in the context of atherosclerosis, including clinical study design and the differential effects of EPA and DHA on cell function. We propose steps to improve clinical and basic experimental study design in order to improve supplement composition optimization. Finally, we propose that understanding the factors underlying the poor response to n-3 PUFAs, and the development of molecular biomarkers for predicting response may help towards a more personalized treatment.

DHA/EPA (Omega-3) and LA/GLA (Omega-6) as Bioactive Molecules in Neurodegenerative Diseases.

Neurodegenerative diseases are characterized by neuroinflammation, neuronal depletion and oxidative stress. They coincide with subtle chronic or flaring inflammation, sometimes escalating with infiltrations of the immune system cells in the inflamed parts causing mild to severe or even lethal damage. Thus, neurodegenerative diseases show all features of autoimmune diseases. Prevalence of neurodegenerative diseases has dramatically increased in recent decades and unfortunately, the therapeutic efficacy and safety profile of available drugs is moderate. The beneficial effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) polyunsaturated fatty acids (omega-3 PUFAs) are nowadays highlighted by a plethora of studies. They play a role in suppression of inflammation, gene expression, cellular membrane fluidity/permeability, immune functionality and intracellular/exocellular signaling. The role of omega-6 polyunsaturated fatty acids, such as linoleic acid (LA), gamma linolenic acid (GLA), and arachidonic acid (AA), on neuroprotection is controversial, as some of these agents, specifically AA, are proinflammatory, whilst current data suggest that they may have neuroprotective properties as well. This review provides an overview of the existing recent clinical studies with respect to the role of omega-3 and omega-6 PUFAs as therapeutic agents in chronic, inflammatory, autoimmune neurodegenerative diseases as well as the dosages and the period used for testing.

Amygdalin as a chemoprotective agent in co-treatment with cisplatin.

Amygdalin is a naturally occurring glycoside used in traditional Chinese medicine and is known to have anti-cancer properties. Even though the anti-cancer properties of amygdalin are well known, its effect on normal cells has not been thoroughly investigated. The aim of the present study was to investigate a possible chemo-protective role of amygdalin against the cytotoxic effects of chemotherapy for normal human cells. Specifically, it was tested in combination with a strong chemotherapeutic drug cisplatin. Human non-tumorigenic MCF12F epithelial cell line, human fibroblasts cells, human breast cancer MCF7 and MDA-MB-231 cells were treated with cisplatin in a dose- and time-depended manner in the absence or presence of amygdalin. When MCF12F cells and fibroblasts underwent pre-treatment with amygdalin followed by cisplatin treatment (24 h amygdalin + 24 h cisplatin), the cell viability was increased (22%, p < 0.001) as indicated using MTT assay. As attested by flow cytometry, combination treatment was associated with decreased the percentage of late apoptotic cells compared with monotherapy (fold-change of decrease = 1.6 and 4.5 for 15 and 20 µΜ, respectively). Also, the proteins expression of PUMA, p53, phospho-p53 and Bax decreased, when a combination treatment was used vs. cisplatin alone, while the proapoptotic proteins Bcl-2 and Bcl-xL exhibited an increased tendency in the presence of amygdalin. Moreover, the levels of pro-apoptotic genes PUMA, p53, and BAX mRNA were significantly downregulated (∼83%, ∼66%, and ∼44%, respectively) vs. cisplatin alone, while the mRNA levels of anti-apoptotic genes BCl-2 and Bcl-XL were upregulated (∼44.5% and ∼51%, respectively), vs. cisplatin alone after 24 h of combination treatment. The study on the Combination index (CI) assay indicated that amygdalin could be possibly considered as an antagonist to cisplatin (2.2 and 2.3) for MCF12F and fibroblast cells, respectively. In contrast, for the breast cancer MCF7 and MDA-MB-231 cells, amygdalin and cisplatin indicated a synergistic effect (0.8 and 0.65), respectively. Our present findings suggest that amygdalin has chemo-modulatory effect when used in co-treatment with cisplatin and is able to protect normal breast cells as well as the fibroblasts during chemotherapy treatment, indicating a strong selective chemoprotective ability and may contribute to a better quality of life for cancer patients.

Natural biomolecules and derivatives as anticancer immunomodulatory agents.

Despite advancements in chemotherapy, the issue of resistance and non-responsiveness to many chemotherapeutic drugs that are currently in clinical use still remains. Recently, cancer immunotherapy has gathered attention as a novel treatment against select cancers. Immunomodulation is also emerging as an effective strategy to improve efficacy. Natural phytochemicals, with known anticancer properties, been reported to mediate their effects by modulating both traditional cancer pathways and immunity. The mechanism of phytochemical mediated-immunomodulatory activity may be attributed to the remodeling of the tumor immunosuppressive microenvironment and the sensitization of the immune system. This allows for improved recognition and targeting of cancer cells by the immune system and synergy with chemotherapeutics. In this review, we will discuss several well-known plant-derived biomolecules and examine their potential as immunomodulators, and therefore, as novel immunotherapies for cancer treatment.

Annonacin promotes selective cancer cell death via NKA-dependent and SERCA-dependent pathways.

In the healthcare sector, phytocompounds are known to be beneficial by contributing or alleviating a variety of diseases. Studies have demonstrated the progressive effects of phytocompounds on immune-related diseases and to exhibit anticancer effects. Graviola tree is an evergreen tree with its extracts (leafs and seeds) been reported having anticancer properties, but the precise target of action is not clear. Using an in silico approach, we predicted that annonacin, an Acetogenin, the active agent found in Graviola leaf extract (GLE) to potentially act as a novel inhibitor of both sodium/potassium (NKA) and sarcoplasmic reticulum (SERCA) ATPase pumps. We were able to validate and confirm the in silico studies by showing that GLE inhibited NKA and SERCA activity in intact cells. In the present study, we also demonstrated the antiproliferative and anticancer effects of GLE in a variety of cancer cell lines with limited toxic effects on non-transformed cells. Moreover, our results revealed that known inhibitors of both NKA and SERCA pumps could also promote cell death in several cancer cell lines. In addition, a mouse xenograft cancer model showed GLE as able to reduce tumor size and progression. Finally, bioprofiling studies indicated a strong correlation between overexpression of both NKA and SERCA gene expression vs. survival rates. Overall, our results demonstrated that GLE can promote selective cancer cell death via inhibiting NKA and SERCA, and thus can be considered as a potential novel treatment for cancer. After molecular analysis of GLE by liquid chromatography-mass spectrometry and ESI-QTOF-MS analysis, it was found that the MS spectrum of the high abundant chromatographic peak purified sample highly consisted of annonacin.

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK.

A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (ß2GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL-ß2GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of ß2GPI in the fluid phase, suggesting that the pathogenic anti-ß2GPI antibodies in this in vitro model target this domain.

Hydroxychloroquine Protects against Cardiac Ischaemia/Reperfusion Injury In Vivo via Enhancement of ERK1/2 Phosphorylation.

An increasing number of investigations including human studies demonstrate that pharmacological ischaemic preconditioning is a viable way to protect the heart from myocardial ischaemia/reperfusion (I/R) injury. This study investigated the role of hydroxychloroquine (HCQ) in the heart during I/R injury. In vitro and in vivo models of myocardial I/R injury were used to assess the effects of HCQ. It was found that HCQ was protective in neonatal rat cardiomyocytes through inhibition of apoptosis, measured by TUNEL and cleaved caspase-3. This protection in vitro was mediated through enhancement of ERK1/2 phosphorylation mediated by HCQ in a dose-dependent fashion. A decrease in infarct size was observed in an in vivo model of myocardial I/R injury in HCQ treated animals and furthermore this protection was blocked in the presence of the ERK1/2 inhibitor U0126. For the first time, we have shown that HCQ promotes a preconditioning like protection in an in vivo simulated rat myocardial I/R injury model. Moreover, it was shown that HCQ is protective via enhanced phosphorylation of the pro-survival kinase ERK1/2.

MultiElec: A MATLAB Based Application for MEA Data Analysis.

We present MultiElec, an open source MATLAB based application for data analysis of microelectrode array (MEA) recordings. MultiElec displays an extremely user-friendly graphic user interface (GUI) that allows the simultaneous display and analysis of voltage traces for 60 electrodes and includes functions for activation-time determination, the production of activation-time heat maps with activation time and isoline display. Furthermore, local conduction velocities are semi-automatically calculated along with their corresponding vector plots. MultiElec allows ad hoc signal suppression, enabling the user to easily and efficiently handle signal artefacts and for incomplete data sets to be analysed. Voltage traces and heat maps can be simply exported for figure production and presentation. In addition, our platform is able to produce 3D videos of signal progression over all 60 electrodes. Functions are controlled entirely by a single GUI with no need for command line input or any understanding of MATLAB code. MultiElec is open source under the terms of the GNU General Public License as published by the Free Software Foundation, version 3. Both the program and source code are available to download from http://www.cancer.manchester.ac.uk/MultiElec/.

Diabetic hearts have lower basal urocortin levels that fail to increase after cardioplegic arrest: association with increased apoptosis and postsurgical cardiac dysfunction.

OBJECTIVES: The present study investigated the cardioprotective role of urocortin (Ucn) and its relationship with protein kinase C (PKC)ε and PKCδ in patients with (DMPs) and without (NDMPs) diabetes mellitus after on-pump cardiac surgery (OPCS). The molecular mechanisms responsible for the reported worse outcomes of DMP after OPCS remain unknown. METHODS: Two sequential biopsy specimens were obtained from the right atrium of 27 DMPs and 22 NDMPs before and after cardiopulmonary bypass. RESULTS: Postcardioplegic induction of Ucn in NDMPs (P<.01) was not observed in the DMPs, whose precardioplegic Ucn levels were 50% lower than those in the NDMPs (P<.05). In the NDMPs, cardioplegic arrest increased PKCε mRNA and protein (P<.05); overexpression of PKCδ was not seen. In contrast, DMPs showed increased PKCδ expression (P<.01), with no change in PKCε. Apoptosis was more than twofold greater in the postcardioplegic samples from the DMPs than in those from the NDMPs. The apoptotic myocytes were Ucn negative and exhibited nuclear relocation of PKCδ. Enhanced PKCε/mitochondrial co-localization was observed in viable, Ucn-positive, myocytes. The leakage of troponin I documented in the DMPs was greater than that in the NDMPs, although the difference was not statistically significant (P=.06). Furthermore, despite a similar incidence of perioperative acute myocardial infarction, the DMPs did not show postoperative improvement of systolic or diastolic function, although that was seen in the NDMPs (P<.05). CONCLUSIONS: Cardioplegic arrest failed to induce in DMPs myocyte overexpression of Ucn or PKCε but was associated with induction and mitochondrial relocation of PKCδ, resulting in apoptosis. Failure to overexpress Ucn in the DMPs was associated with apoptosis and cardiac dysfunction and, thus, might contribute to worse postoperative outcomes.

The role and modulation of autophagy in experimental models of myocardial ischemia-reperfusion injury.

A physiological sequence called autophagy qualitatively determines cellular viability by removing protein aggregates and damaged cytoplasmic constituents, and contributes significantly to the degree of myocardial ischemia-reperfusion (I/R) injury. This tightly orchestrated catabolic cellular 'housekeeping' process provides cells with a new source of energy to adapt to stressful conditions. This process was first described as a pro-survival mechanism, but increasing evidence suggests that it can also lead to the demise of the cell. Autophagy has been implicated in the pathogenesis of multiple cardiac conditions including myocardial I/R injury. However, a debate persists as to whether autophagy acts as a protective mechanism or contributes to the injurious effects of I/R injury in the heart. This controversy may stem from several factors including the variability in the experimental models and species, and the methodology used to assess autophagy. This review provides updated knowledge on the modulation and role of autophagy in isolated cardiac cells subjected to I/R, and the growing interest towards manipulating autophagy to increase the survival of cardiac myocytes under conditions of stress-most notably being I/R injury. Perturbation of this evolutionarily conserved intracellular cleansing autophagy mechanism, by targeted modulation through, among others, mammalian target of rapamycin (mTOR) inhibitors, adenosine monophosphate-activated protein kinase (AMPK) modulators, calcium lowering agents, resveratrol, longevinex, sirtuin activators, the proapoptotic gene Bnip3, IP3 and lysosome inhibitors, may confer resistance to heart cells against I/R induced cell death. Thus, therapeutic manipulation of autophagy in the challenged myocardium may benefit post-infarction cardiac healing and remodeling.

Cardiac tissue engineering: renewing the arsenal for the battle against heart disease.

The development of therapies that lead to the regeneration or functional repair of compromised cardiac tissue is the most important challenge facing translational cardiovascular research today. During the last 25 years huge efforts have been made towards restoring the physiologic functions of the heart by means of delivering cell implants into the insulted heart, initially through 'naked cell' injections and more recently through the principle of cardiac tissue engineering and the use of elaborate delivery systems and priming mechanisms that include scaffolds, bioreactors or ex vivo manipulations of cells and support structures. In this review we summarise various approaches towards cardiac repair and highlight advances in the field of tissue engineering, ranging from a review of cell types used, to advances that attempt to address mechanistic and functional elements that are critical for successful restoration of the heart, including the maintenance of the extracellular matrix through scaffoldless cardiac sheets, strategies that promote neovascularisation and the precise micro-delivery of cell populations to form three-dimensional structures through bioengineering methods such as microfabrication.

The cardioprotective effects of urocortin are mediated via activation of the Src tyrosine kinase-STAT3 pathway.

Src tyrosine kinase family was recently identified as a novel upstream modulator of MAP kinase subfamily, p42/p44, whose activation is required for urocortin (Ucn)-mediated cardioprotection. Src kinase was also shown to reduce apoptosis in different cancer cell lines, enhancing phosphorylation and DNA binding affinity of signal transducer and activator of transcription (STAT)3. In order to evaluate the effects of Ucn on the activation status of different STAT family members, HL-1 cardiac cells were incubated with Ucn (10 nM) for increasing periods of time. STAT3 was rapidly phosphorylated at Tyr705, while neither phosphorylation at Ser727 nor induction of total STAT3 was observed. Pretreatment with PP2, a selective inhibitor of Src tyrosine kinase, reduced the pSTAT(-T705) phosphorylation and transcriptional activity induced by Ucn in a dose-dependent manner. Overexpression of STAT3 in HL-1 cardiac myocytes pretreated with Ucn reduced the magnitude of cell death as compared with Ucn treatment alone, while transfection of HL-1 cells with a STAT3 mutant functionally inactive, acting as a dominant negative (DN-STAT3), enhanced the extent of cell death in a dose-dependent manner. In line with this finding, in HL-1 cardiac myocytes overexpressing STAT3 treated with Ucn, addition of the Src kinase inhibitor PP2 reversed the cytoprotective effects of Ucn, proving that the cytoprotective effects of Ucn are also mediated via the Src-pSTAT(-T705) phosphorylation pathway. By immunocytochemistry, Ucn induced nuclear translocation of pST3-T705, which was inhibited by pretreatment with PP2. Together, these data strongly suggest that Ucn can mediate cardioprotection by activating the Src-pSTAT-T705 phosphorylation pathway.

Targeting urocortin signaling pathways to enhance cardioprotection: is it time to move from bench to bedside?

Despite the exponential growth in medical knowledge, cardiovascular diseases (CVDs) contribute to more than one-third of worldwide morbidity and mortality. A range of therapies already exist for established CVDs, although there is significant interest in further understanding their pathogenesis. The urocortins (Ucns) are peptide members of the corticotrophin-releasing factor family, a group of evolutionary conserved peptides with homologues in fish, amphibians and mammals and considered to play a pivotal role in energy homeostasis and local tissue repair. A number of preclinical studies in vitro, in-vivo and ex-vivo have defined a multifaceted effect of Ucns on the cardiovascular system. Different G-protein coupled signaling and protein-kinase pathways have been shown to be activated by Ucns, together with different transcriptional and translational effects, all of which preferentially converge on the mitochondria, where the modulation of apoptosis is considered their principal action. It has been demonstrated in experimental models, and consequentially suggested in human diseases, that Ucn-mediated inhibition of apoptosis can be exploited for the improvement of both therapeutic and preventative strategies against CVDs. Specifically, some unavoidable iatrogenic ischemia/reperfusion (I/R) injuries, e.g. during cardiac surgery or percutaneous coronary angioplasty, may greatly benefit from the anti-apoptotic effect of Ucns. However, few studies on the topic have been employed in humans to date. Therefore, this review will focus on the different intra-cellular mechanisms of action of Urocortins, and detail the different Ucn-mediated pathways identified so far. It will also highlight the limited evidence already existing in human clinical and surgical settings, as well as emphasize the potential uses of Ucns in human cardiac pathology.