Cyclophilin B serum levels present variations across the menstrual cycle.

Cyclophilins are a family of chaperones involved in inflammation and cell death. Cyclophilin B is released by inflammatory cells and acts through the receptor CD147, affecting matrix metalloproteases release, whilst cyclophilin D participates in hypoxia-induced apoptosis. Previous studies related hormones like estradiol or prolactin to these proteins, however, their blood concentrations across the menstrual cycle have not been determined. In this work, eleven healthy women (BMI: 21.8 kg/m2) were monitored during a single menstrual cycle, making blood extractions at follicular, periovulatory and mid-luteal phases. Hormone and cyclophilin levels were determined in each phase. Statistical differences were determined by repeated measures ANOVA and estimated marginal means tests, or by Friedman and Dunn-Bonferroni tests for parametric and non-parametric variables, respectively. Bivariate correlations were evaluated with the Spearman coefficient. Cyclophilin B concentrations presented significant differences during the menstrual cycle (p = 0.012). The highest levels of this protein were found at follicular extraction, followed by a decrease at periovulatory phase and a slight increase at mid-luteal phase. Cyclophilin D showed the same profile, although statistical significance was not reached. This immunophilin exhibited a positive correlation with luteinizing hormone at periovulatory phase (r = 0.743, p = 0.009) and with follicle stimulating hormone at mid-luteal phase (r = 0.633, p = 0.036). This is the first study describing the changes in cyclophilin B concentrations across the menstrual cycle, as well as the association of luteinizing and follicle stimulating hormones with cyclophilin D. These results suggest a role of these proteins in the cyclic inflammatory events that affect female reproductive system that should be explored.

Cyclophilins modify their profile depending on the organ or tissue in a murine inflammatory model.

Inflammation is the leading subjacent cause of many chronic diseases. Despite several studies in the last decades, the molecular mechanism involving its pathophysiology is not fully known. Recently, the implication of cyclophilins in inflammatory-based diseases has been demonstrated. However, the main role of cyclophilins in these processes remains elusive. Hence, a mouse model of systemic inflammation was used to better understand the relationship between cyclophilins and their tissue distribution. To induce inflammation, mice were fed with high-fat diet for 10 weeks. In these conditions, serum levels of interleukins 2 and 6, tumour necrosis factor-α, interferon-ϒ, and the monocyte chemoattractant protein 1 were elevated, evidencing a systemic inflammatory state. Then, in this inflammatory model, cyclophilins and CD147 profiles in the aorta, liver, and kidney were studied. The results demonstrate that, upon inflammatory conditions, cyclophilins A and C expression levels were increased in the aorta. Cyclophilins A and D were augmented in the liver, meanwhile, cyclophilins B and C were diminished. In the kidney, cyclophilins B and C levels were elevated. Furthermore, CD147 receptor was also increased in the aorta, liver, and kidney. In addition, when cyclophilin A was modulated, serum levels of inflammatory mediators were decreased, indicating a reduction in systemic inflammation. Besides, the expression levels of cyclophilin A and CD147 were also reduced in the aorta and liver, when cyclophilin A was modulated. Therefore, these results suggest that each cyclophilin has a different profile depending on the tissue, under inflammatory conditions.

Association of cyclophilins and cardiovascular risk factors in coronary artery disease.

Cyclophilins are chaperone proteins that play important roles in signal transduction. Among them, cyclophilins A, B, C, and D were widely associated with inflammation and cardiovascular diseases. Cyclophilins A and C have been proposed as coronary artery disease biomarkers. However, less is known about their relationship with cardiovascular risk factors. Therefore, this study aimed to determine the association between cyclophilin A, B, C, and D and cardiovascular risk factors in coronary artery disease. Serum levels of cyclophilins were measured in 167 subjects (subdivided according to cardiovascular risk factors presence). This study reveals that cyclophilin A and C are elevated in patients regardless of the risk factors presence. Moreover, cyclophilin B is elevated in male patients with hypertension, type 2 diabetes, or high glucose levels. In addition, cyclophilins A, B, and C were significantly correlated with cardiovascular risk factors, but only cyclophilin B was associated with type 2 diabetes. The multivariate analysis strengthens the predictive value for coronary artery disease presence of cyclophilin A (>8.2 ng/mL) and cyclophilin C (>17.5 pg/mL) along with the cardiovascular risk factors tobacco, hypertension, dyslipidemia, and high glucose and cholesterol levels. Moreover, the risk of coronary artery disease is increased in presence of cyclophilin B levels above 63.26 pg/mL and with hypertension or dyslipidemia in male patients. Consequently, cyclophilins A and C serum levels are reinforced as useful coronary artery disease biomarkers, meanwhile, cyclophilin B is a valuable biomarker in the male population when patients are also suffering from hypertension or dyslipidemia.

Extracellular cyclophilins A and C induce dysfunction of pancreatic microendothelial cells.

Extracellular cyclophilins (eCyps) A and B are chemotactic mediators in several illnesses in which inflammation plays an important role such as diabetes and cardiovascular diseases. Recently, eCypC has been reported as a potential biomarker for coronary artery disease but its effect in endothelium has not been determined. Moreover, there is a lack of studies with all these proteins in the same model, which makes difficult a direct comparison of their effects. In this work, MS1 pancreatic microendothelial cells were treated with eCyps A, B and C and their impact on endothelial function was analysed. eCyps A and C stimulated the release of IL-6 and MCP-1 and increased the expression of the receptor CD147, but eCypB did not affect these pro-inflammatory markers. Moreover, eCypC activated the translocation of NFkB-p65 to the nucleus. All these effects were reversed by pre-treatment with cyclosporine A. eCyps also produced endothelial dysfunction, as evidenced by the decrease in eNOS activation. Finally, the crosstalk among eCyps addition and their protein and gene expression was evaluated. eCypA generated a depletion in its protein and gene levels, whilst eCyps B and C upregulated their own protein expression. Moreover, each eCyp altered the intracellular expression of other Cyps, including cyclophilin D. This work is the first report of eCyps influence on iCyps expression, as well as the first description of eCypC as an activator of CD147 receptor and a mediator of endothelial dysfunction, which points to a potential role of this protein in vascular complications associated to diabetes.

Enniatins A1 and B1 alter calcium homeostasis of neuronal cells leading to apoptotic death.

Enniatins (ENNs) A1 and B1 are non-regulated mycotoxins produced by Fusarium spp. that commonly occur in different types of food. These toxins are cytotoxic in several cell lines, but their mechanism of action is unclear. In this study, the cytotoxic effects of ENNs A1 and B1 in SH-SY5Y human neuroblastoma cells were analysed. Moreover, to better understand their mechanism of action, mitochondrial function, reactive oxygen species (ROS) levels and calcium fluxes were monitored. ENNs A1 and B1 reduced cell viability, presenting IC50 values of 2.0 and 2.7 µM, respectively. Both toxins induced caspase-dependent apoptosis, but only ENN A1 increased ROS production. Apoptotic cell death seems to be triggered by the increase in cytosolic calcium produced by both ENNs, since the toxins altered Ca2+ homeostasis by depleting intracellular reservoirs. Finally, binary combinations of ENN A1, ENN B1, ENN A and ENN B were tested. All mixtures resulted in an antagonistic effect, with the exception of ENN A and ENN B1 combination, which produced an additive effect. The results presented in this study provide the first evidence of ENNs A1 and B1 effects on calcium fluxes, providing new insights into the mechanism of action of these mycotoxins.

Furanoditerpenes from Spongia (Spongia) tubulifera Display Mitochondrial-Mediated Neuroprotective Effects by Targeting Cyclophilin D.

Neuroprotective properties of five previously described furanoditerpenes 1-5, isolated from Spongia (Spongia) tubulifera, were evaluated in an in vitro oxidative stress model in SH-SY5Y cells. Dose-response treatments revealed that 1-5 improved cell survival at nanomolar concentrations through the restoration of mitochondrial membrane potential and the reduction of reactive oxygen species. Their ability to prevent the mitochondrial permeability transition pore opening was also assessed, finding that 4 and 5 inhibited the channel at 0.001 µM. This inhibition was accompanied by a decrease in the expression of cyclophilin D, the main regulator of the pore, which was also reduced by 1 and 2. However, the activation of ERK and GSK3ß, upstream modulators of the channel, was not affected by compounds. Therefore, their ability to bind cyclophilin D was evaluated by surface plasmon resonance, observing that 2-5 presented equilibrium dissociation constants in the micromolar range. All compounds also showed affinity for cyclophilin A, being 1 selective toward this isoform, while 2 and 5 exhibited selectivity for cyclophilin D. When the effects on the intracellular expression of cyclophilins A-C were determined, it was found that only 1 decreased cyclophilin A, while cyclophilins B and C were diminished by most compounds, displaying enhanced effects under oxidative stress conditions. Results indicate that furanoditerpenes 1-5 have mitochondrial-mediated neuroprotective properties through direct interaction with cyclophilin D. Due to the important role of this protein in oxidative stress and inflammation, compounds are promising drugs for new therapeutic strategies against neurodegeneration.

Single and combined effects of regulated and emerging mycotoxins on viability and mitochondrial function of SH-SY5Y cells.

Co-occurrence of emerging and regulated mycotoxins in contaminated samples has been widely documented, but studies about their combined toxicity are scarce. In this report, the regulated mycotoxins deoxynivalenol, fumonisin B1 and zearalenone, and the emerging ones enniatin A, enniatin B and beauvericin were tested in SH-SY5Y human neuroblastoma cells. Their individual and binary combined effects on cell viability and mitochondrial function were evaluated. The results with individual mycotoxins revealed that deoxynivalenol and emerging mycotoxins were the most damaging to neuronal cells, presenting IC50 values between 0.35 and 2.4 µM. Interestingly, non-regulated mycotoxins triggered apoptosis by affecting to mitochondrial membrane potential. However, when regulated and non-regulated mycotoxins were binary mixed, antagonistic effects were found in all cases. Finally, cow feed and milk extracts were analysed by UHPLC-MS/MS, detecting the presence of several mycotoxins included in this study. These extracts were tested in neuroblastoma cells, and damaging effects on cell viability were found. Although binary combinations of mycotoxins produced antagonistic effects, their mixture in natural matrixes induces greater effects than expected. Therefore, it would be interesting to explore the matrix influence on mycotoxin toxicity, and to continue studying the neurotoxic mechanism of action of emerging mycotoxins, as they could be a health hazard.

Anhydroexfoliamycin, a Streptomyces Secondary Metabolite, Mitigates Microglia-Driven Inflammation.

Anhydroexfoliamycin, a secondary metabolite from Streptomyces, has shown antioxidant properties in primary cortical neurons reducing neurodegenerative hallmarks diseases, both in vitro and in vivo models. Activated microglia, in the central nervous system, plays a crucial role in neuroinflammation and is associated with neurodegeneration. Therefore, the aim of the present study was to determine the anti-inflammatory and antioxidant potential of the anhydroexfoliamycin over microglia BV2 cells. Neuroinflammation was simulated by incubation of microglia cells in the presence of lipopolysaccharide to activate proinflammatory transduction pathways. Moreover, a coculture of neuron SH-SY5Y and microglia BV2 cells was used to evaluate the neuroprotective properties of the Streptomyces metabolite. When microglia cells were preincubated with anhydroexfoliamycin, proinflammatory pathways, such as the translocation of the nuclear factor κB, the phosphorylation of c-Jun N-terminal kinase, and the inducible nitric oxide synthase expression, were inhibited. In addition, intracellular reactive oxygen species generation and the liberation of nitric oxide, interleukin 6, and tumor necrosis factor α were also decreased. Besides, the Streptomyces-derived compound showed antioxidant properties promoting the translocation of the factor erythroid 2-related factor 2 and protecting the SH-SY5Y cells from the neurotoxic mediators released by activated microglia. The effects of this compound were at the same level as the immunosuppressive drug cyclosporine A. Therefore, these results indicate that anhydroexfoliamycin is a promising tool to control microglia-driven inflammation with therapeutic potential in neuroinflammation.

Cyclophilins A, B, and C Role in Human T Lymphocytes Upon Inflammatory Conditions.

Cyclophilins (Cyps) are a group of peptidyl-prolyl cis/trans isomerases that play crucial roles in regulatory mechanisms of cellular physiology and pathology in several inflammatory conditions. Their receptor, CD147, also participates in the development and progression of the inflammatory response. Nevertheless, the main function of Cyps and their receptor are yet to be deciphered. The release of CypA and the expression of the CD147 receptor in activated T lymphocytes were already described, however, no data are available about other Cyps in these cells. Therefore, in the present work intra and extracellular CypA, B and C levels were measured followed by induced inflammatory conditions. After activation of T lymphocytes by incubation with concanavalin A, both intra and extracellular Cyps levels and the CD147 membrane receptor expression were increased leading to cell migration towards circulating CypA and CypB as chemoattractants. When CypA was modulated by natural and synthetic compounds, the inflammatory cascade was avoided including T cell migration. Our results strengthen the relationship between CypA, B, and C, their receptor, and the inflammatory process in human T lymphocytes, associating CypC with these cells for the first time.

Crosstalk between cyclophilins and T lymphocytes in coronary artery disease.

Cardiovascular diseases and atherosclerosis are currently some of the most widespread diseases of our time. Within cardiovascular disease, coronary artery disease and underlying atherosclerosis were recently linked with systemic and local inflammation. Cyclophilins participate in the initiation and progression of these inflammatory-related diseases. Cyclophilins are released into the extracellular space upon inflammatory stimuli and participate in the pathology of cardiovascular diseases. The cell surface receptor for extracellular cyclophilins, the CD147 receptor, also contributes to coronary artery disease pathogenesis. Nevertheless, the physiological relevance of cyclophilin's family and their receptor in cardiovascular diseases remains unclear. The present study aimed to better understand the role of cyclophilins in cardiovascular artery disease and their relationship with inflammation. Hence, cyclophilins and pro-inflammatory interleukins were measured in the serum of 30 subjects (divided into three groups according to coronary artery disease status: 10 patients with acute coronary syndrome, 10 patients with chronic coronary artery disease, and 10 control volunteers). In addition, cyclophilin levels and CD147 receptor expression were measured in T lymphocytes purified from these subjects. Cyclophilin A, B, and C, pro-inflammatory interleukins, and CD147 membrane expression were significantly elevated in patients with coronary artery disease.

Cyclophilins in Ischemic Heart Disease: Differences Between Acute and Chronic Coronary Artery Disease Patients.

BACKGROUND: Cyclophilins (Cyps) are a family of peptidyl-prolyl cis/trans isomerases consistently involved in cardiovascular diseases through the inflammation pathway. This study aims to investigate the serum levels of Cyps (CypA, CypB, CypC and CypD) in patients with coronary artery disease (CAD) and the correlation with clinical characteristics and inflammation parameters. METHODS: We developed an observational prospective study with a total of 125 subjects: 40 patients with acute CAD, 40 patients with chronic CAD and 45 control volunteers, in whom serum levels of Cyps (CypA, CypB, CypC and CypD), interleukins and metalloproteinases were measured. RESULTS: CypA levels increased significantly in CAD patients compared with control subjects, but no differences were noted between acute CAD (7.80 ± 1.30 ng/mL) and chronic CAD (5.52 ± 0.76 ng/mL) patients (P = 0.13). No differences in CypB and CypD levels were showed between CAD patients and controls and between acute CAD and chronic CAD patients. In relation with CypC, the levels in CAD patients were significantly higher compared to controls (32.42 ± 3.71 pg/mL vs. 9.38 ± 1.51 pg/mL, P < 0.001), but no differences between acute and chronic CAD groups were obtained (P = 0.62). We analyzed the CypC > 17.5 pg/mL cut-off point, and it was significantly associated with older age, hypertension, dyslipidemia and more extensive CAD in acute and chronic CAD groups. CONCLUSIONS: CypA and CypC levels are increased in CAD patients. High CypC serum levels could be a novel biomarker in CAD patients correlating with a more severe disease.

Futunamine, a Pyrrole-Imidazole Alkaloid from the Sponge Stylissa aff. carteri Collected off the Futuna Islands.

The chemical investigation of the sponge Stylissa aff. carteri collected around Futuna Islands in the Pacific Ocean led to the isolation of three new dimeric pyrrole 2-aminoimidazole alkaloids (PIAs). Futunamine (1) features an unprecedented pyrrolo[1,2-c]imidazole core, while two other new dimeric PIAs were identified as analogues of palau'amine. Together with other known PIAs isolated from this species, they were shown to exhibit anti-inflammatory and neuroprotective activities.

Gracilin-Derivatives as Lead Compounds for Anti-inflammatory Effects.

Gracilins are diterpenes derivative, isolated from the marine sponge Spongionella gracilis. Natural gracilins and synthetic derivatives have shown antioxidant, immunosuppressive, and neuroprotective capacities related to the affinity for cyclophilins. The aim of this work was to study anti-inflammatory and immunosuppressive pathways modulated by gracilin L and two synthetic analogues, compound 1 and 2, on a cellular model of inflammation. In this way, the murine BV2 microglia cell line was used. To carry out the experiments, microglia cells were pre-treated with compounds for 1 h and then stimulated with lipopolysaccharide for 24 h to determine reactive oxygen species production, mitochondrial membrane potential, the release of nitric oxide, interleukin-6 and tumor necrosis factor-α and the expression of Nuclear factor-erythroid 2-related factor 2, Nuclear Factor-κB, the inducible nitric oxide synthase, and the cyclophilin A. Finally, a co-culture of neuron SH-SY5Y and microglia BV2 cells was used to check the neuroprotective effect of these compounds. Cyclosporine A was used as a control of effect. The compounds were able to decrease inflammatory mediators, the expression of inflammatory target proteins as well as they activated anti-oxidative mechanism upon inflammatory conditions. For this reason, natural and synthetic gracilins could be interesting for developing anti-inflammatory drugs.

High Serum Cyclophilin C levels as a risk factor marker for Coronary Artery Disease.

Cyclophilins (Cyps) are ubiquitous proteins that belong to the immunophilins family consistently associated with inflammatory and cardiovascular diseases. While levels of CypA have been extensively studied, less data are available for other Cyps. The purpose of this case-control study was to determine the relationship of Cyps (A, B, C and D) with coronary artery disease (CAD) and eight inflammation markers. Serum levels of Cyps, interleukins and metalloproteinases were measured in serum collected from 84 subjects. Participants were divided into two sub-groups based on CAD diagnosis: 40 CAD patients and 44 control volunteers. Serum levels of CypA, CypB and CypC, IL-1ß and IL-6 were significantly higher in CAD patients. Bivariate correlation analysis revealed a significant positive correlation between Cyps and several blood and biochemical parameters. When the ability of Cyps levels for CAD diagnosis was evaluated, higher sensitivity and selectivity values were obtained with CypC (c-statistic 0.891, p < 0.001) indicating that it is a good marker of CAD disease, while less conclusive results were obtained with CypA (c-statistic 0.748, p < 0.001) and CypB (c-statistic 0.655, p < 0.014). In addition, significant correlations of traditional CAD risk factors and CypC were observed. In summary, high levels of CypC are a risk factor for CAD and therefore it can be proposed as a new biomarker for this disease.

Bromotryptamine and Bromotyramine Derivatives from the Tropical Southwestern Pacific Sponge Narrabeena nigra.

So far, the Futuna Islands located in the Central Indo-Pacific Ocean have not been inventoried for their diversity in marine sponges and associated chemical diversity. As part of the Tara Pacific expedition, the first chemical investigation of the sponge Narrabeena nigra collected around the Futuna Islands yielded 18 brominated alkaloids: seven new bromotryptamine derivatives 1-7 and one new bromotyramine derivative 8 together with 10 known metabolites of both families 9-18. Their structures were deduced from extensive analyses of nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) data. In silico metabolite anticipation using the online tool MetWork revealed the presence of a key and minor biosynthetic intermediates. These 18 compounds showed almost no cytotoxic effect up to 10 µM on human neuroblastoma SH-SY5Y and microglia BV2 cells, and some of them exhibited an interesting neuroprotective activity by reducing oxidative damage.

Treasures from the Deep: Characellides as Anti-Inflammatory Lipoglycotripeptides from the Sponge Characella pachastrelloides.

The chemical investigation of marine invertebrates from the deep Northeastern Atlantic revealed new lipoglycotripeptides named characellides isolated from the tetractinellid sponge Characella pachastrelloides. This new family of natural products features a central tripeptide linked to a rare sugar unit and a long alkyl chain ending with a 2,3-dimethyltetrahydropyran. The configurations of all 13 chiral centers were determined by extensive use of NMR data and circular dichroism spectra combined with calculations.

Zoanthamine Alkaloids from the Zoantharian Zoanthus cf. pulchellus and Their Effects in Neuroinflammation.

Two new zoanthamine alkaloids, namely 3-acetoxynorzoanthamine (1) and 3-acetoxyzoanthamine (2), have been isolated from the zoantharian Zoanthus cf. pulchellus collected off the coast of the Santa Elena Peninsula, Ecuador, together with three known derivatives: zoanthamine, norzoanthamine, and 3-hydroxynorzoanthamine. The chemical structures of 1 and 2 were determined by interpretation of their 1D and 2D NMR data and comparison with literature data. This is the first report of zoanthamine-type alkaloids from Zoanthus cf. pulchellus collected in the Tropical Eastern Pacific. The neuroinflammatory activity of all the isolated compounds was evaluated in microglia BV-2 cells and high inhibitory effects were observed in reactive oxygen species (ROS) and nitric oxide (NO) generation.

Evaluation of the Protective Effects of Sarains on H2O2-Induced Mitochondrial Dysfunction and Oxidative Stress in SH-SY5Y Neuroblastoma Cells.

Sarains are diamide alkaloids isolated from the Mediterranean sponge Haliclona (Rhizoniera) sarai that have previously shown antibacterial, insecticidal and anti-fouling activities. In this study, we examined for the first time the neuroprotective effects of sarains 1, 2 and A against oxidative stress in a human neuronal model. SH-SY5Y cells were co-incubated with sarains at concentrations ranging from 0.01 to 10 µM, and the well-known oxidant hydrogen peroxide at 150 µM for 6 h and the protective effects of the compounds were evaluated. Among the sarains tested, sarain A was the most promising compound, improving mitochondrial function and decreasing reactive oxygen species levels in human neuroblastoma cells treated with the compound at 0.01, 0.1 and 1 µM. This compound was also able to increase the activity of the antioxidant enzymes superoxide dismutases by inducing the translocation of the nuclear factor E2-related factor 2 (Nrf2) to the nucleus at the lower concentrations tested (0.01 and 0.1 µM). Moreover, sarain A at 0.1 and 1 µM blocked the mitochondrial permeability transition pore (mPTP) opening through cyclophilin D inhibition. These results suggest that the protective effects produced by the treatment with sarain A are related with its ability to block the mPTP and to enhance the Nrf2 pathway, indicating that sarain A may be a candidate compound for further studies in neurodegenerative diseases.

Tetracyclic Truncated Analogue of the Marine Toxin Gambierol Modifies NMDA, Tau, and Amyloid ß Expression in Mice Brains: Implications in AD Pathology.

Gambierol and its two, tetra- and heptacyclic, analogues have been previously proved as promising molecules for the modulation of Alzheimer's disease (AD) hallmarks in primary cortical neurons of 3xTg-AD fetuses. In this work, the effect of the tetracyclic analogue of gambierol was tested in vivo in 3xTg-AD mice (10 months old) after 1 month of weekly treatment with 50 µg/kg. Adverse effects were not reported throughout the whole treatment period and no pathological signs were observed for the analyzed organs. The compound was found in brain samples after intraperitoneal injection. The tetracyclic analogue of gambierol elicited a decrease of amyloid ß1-42 levels and a dose-dependent inhibition of ß-secretase enzyme-1 activity. Moreover, this compound also reduced the phosphorylation of tau at the 181 and 159/163 residues with an increase of the inactive isoform of the glycogen synthase kinase-3ß. In accordance with our in vitro neuronal model, this compound produced a reduction in the N2A subunit of the N-methyl-d-aspartate (NMDA) receptor. The combined effect of this compound on amyloid ß1-42 and tau phosphorylation represents a multitarget therapeutic approach for AD which might be more effective for this multifactorial and complex neurodegenerative disease than the current treatments.