The Role of Calcium-Sensing Receptors in Endothelin-1-Dependent Effects on Adult Rat Ventricular Cardiomyocytes: Possible Contribution to Adaptive Myocardial Hypertrophy.

Nitric oxide (NO)-deficiency as it occurs during endothelial dysfunction activates the endothelin-1 (ET-1) system and increases the expression of receptor activity modifying protein (RAMP)-1 that acts as a chaperon for calcium-sensing receptors (CaR) that have recently been identified to improve cardiac function. Here, we hypothesized that ET-1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP-1, endothelin receptors, and CaR were analyzed by RT-PCR in left ventricular tissues of L-NAME-treated rats. Effects of ET-1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP-1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1 , ETB2 , and ETA receptors. Non-specific NO synthase inhibition with L-Nitro arginine methyl ester (L-NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of ET-1 on cardiomyocytes. Indeed, ET-1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions, cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Inhibition of ETA and ETB1 receptors led to ET-1-dependent reduction in cell shortening and attenuated up-regulation of CaR. Down-regulation of RAMP-1 reduced CaR responsiveness. In conclusion, ET-1 causes an adaptive type of hypertrophy by up-regulation of CaR in cardiomyocytes via ETA and/or ETB1 receptors. J. Cell. Physiol. 232: 2508-2518, 2017. © 2016 Wiley Periodicals, Inc.

12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction.

To understand the mechanisms of 15(S)-HETE-induced endothelial cell (EC) barrier dysfunction, we examined the role of xanthine oxidase (XO). 15(S)-HETE induced junction adhesion molecule A (JamA) phosphorylation on Y164, Y218, and Y280 involving XO-mediated reactive oxygen species production and Src and Pyk2 activation, resulting in its dissociation from occludin, thereby causing tight junction (TJ) disruption, increased vascular permeability, and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on Y164, Y218, and Y280 appears to be critical for its role in 15(S)-HETE-induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high-fat diet (HFD) feeding, WT, but not 12/15-lipoxygenase (LO)(-/-), mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by allopurinol. These observations provide novel insights on the role of XO in 12/15-LO-induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD.

Vascular endothelial tight junctions and barrier function are disrupted by 15(S)-hydroxyeicosatetraenoic acid partly via protein kinase C ε-mediated zona occludens-1 phosphorylation at threonine 770/772.

Disruption of tight junctions (TJs) perturbs endothelial barrier function and promotes inflammation. Previously, we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid, by stimulating zona occludens (ZO)-2 tyrosine phosphorylation and its dissociation from claudins 1/5, induces endothelial TJ disruption and its barrier dysfunction. Here, we have studied the role of serine/threonine phosphorylation of TJ proteins in 15(S)-HETE-induced endothelial TJ disruption and its barrier dysfunction. We found that 15(S)-HETE enhances ZO-1 phosphorylation at Thr-770/772 residues via PKCε-mediated MEK1-ERK1/2 activation, causing ZO-1 dissociation from occludin, disrupting endothelial TJs and its barrier function, and promoting monocyte transmigration; these effects were reversed by T770A/T772A mutations. In the arteries of WT mice ex vivo, 15(S)-HETE also induced ZO-1 phosphorylation and endothelial TJ disruption in a PKCε and MEK1-ERK1/2-dependent manner. In line with these observations, in WT mice high fat diet feeding induced 12/15-lipoxygenase (12/15-LO) expression in the endothelium and caused disruption of its TJs and barrier function. However, in 12/15-LO(-/-) mice, high fat diet feeding did not cause disruption of endothelial TJs and barrier function. These observations suggest that the 12/15-LO-12/15(S)-HETE axis, in addition to tyrosine phosphorylation of ZO-2, also stimulates threonine phosphorylation of ZO-1 in the mediation of endothelial TJ disruption and its barrier dysfunction.

Mechanisms by which calcium receptor stimulation modifies electromechanical coupling in isolated ventricular cardiomyocytes.

The calcium-sensing receptor (CaR) is widely expressed throughout the entire cardiovascular system and is capable of activating signaling pathways in different cells. Alongside calcium, the CaR also responds to physiological polycations such as putrescine underlining a participation in physiological and pathophysiological processes. Here, we aimed to determine mechanisms as to how CaR activation affects the contractile responsiveness of ventricular cardiomyocytes under basal and stimulated conditions. For that purpose, cardiac myocytes from 3-month-old male Wistar rats were isolated, and the acute effects of an antagonist (NPS2390), agonists (putrescine and gadolinium), or of downregulation of the CaR by siRNA on cell shortening were recorded in a cell-edge-detection system. In addition, experiments were performed on muscle stripes and Langendorff preparations. Mechanistic insights were taken from calcium transients of beating fura-2 AM-loaded cardiomyocytes and western blots. Isolated ventricular cardiomyocytes constitutively express CaR. The expression in the atria is less pronounced. Acute inhibition of CaR reduced basal cell shortening of ventricular myocytes at nearly physiological levels of extracellular calcium. Inhibition of CaR strongly reduced contractility of ventricular muscle stripes but not of atria. Activation of CaR by putrescine and gadolinium influences the contractile responsiveness of isolated cardiomyocytes. Increased calcium mobilization from the sarcoplasmic reticulum via an IP3-dependent mechanism was responsible for amplified systolic calcium transients and a subsequent improvement in cell shortening. Alongside with these effects, activation of CaR increased relaxation velocity of the cells. In conclusion, ventricular CaR expression affects contractile parameters of ventricular heart muscle cells and modifies electromechanical coupling of cardiomyocytes.

12/15-Lipoxygenase mediates high-fat diet-induced endothelial tight junction disruption and monocyte transmigration: a new role for 15(S)-hydroxyeicosatetraenoic acid in endothelial cell dysfunction.

A convincing body of evidence suggests that 12/15-lipoxygenase (12/15-LO) plays a role in atherosclerosis. However, the mechanisms of its involvement in the pathogenesis of this disease are not clear. Therefore, the purpose of this study is to understand the mechanisms by which 12/15-LO mediates endothelial dysfunction. 15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 12/15-LO metabolite of arachidonic acid (AA), induced endothelial barrier permeability via Src and Pyk2-dependent zonula occluden (ZO)-2 tyrosine phosphorylation and its dissociation from the tight junction complexes. 15(S)-HETE also stimulated macrophage adhesion to the endothelial monolayer in Src and Pyk2-dependent manner. Ex vivo studies revealed that exposure of arteries from WT mice to AA or 15(S)-HETE led to Src-Pyk2-dependent ZO-2 tyrosine phosphorylation, tight junction disruption, and macrophage adhesion, whereas the arteries from 12/15-LO knock-out mice are protected from these effects of AA. Feeding WT mice with a high-fat diet induced the expression of 12/15-LO in the arteries leading to tight junction disruption and macrophage adhesion and deletion of the 12/15-LO gene disallowed these effects. Thus, the findings of this study provide the first evidence of the role of 12/15-LO and its AA metabolite, 15(S)-HETE, in high-fat diet-induced endothelial tight junction disruption and macrophage adhesion, the crucial events underlying the pathogenesis of atherosclerosis.

Endothelial PlexinD1 signaling instructs spinal cord vascularization and motor neuron development.

How the vascular and neural compartment cooperate to achieve such a complex and highly specialized structure as the central nervous system is still unclear. Here, we reveal a crosstalk between motor neurons (MNs) and endothelial cells (ECs), necessary for the coordinated development of MNs. By analyzing cell-to-cell interaction profiles of the mouse developing spinal cord, we uncovered semaphorin 3C (Sema3C) and PlexinD1 as a communication axis between MNs and ECs. Using cell-specific knockout mice and in vitro assays, we demonstrate that removal of Sema3C in MNs, or its receptor PlexinD1 in ECs, results in premature and aberrant vascularization of MN columns. Those vascular defects impair MN axon exit from the spinal cord. Impaired PlexinD1 signaling in ECs also causes MN maturation defects at later stages. This study highlights the importance of a timely and spatially controlled communication between MNs and ECs for proper spinal cord development.

X-Aptamer Technology Identifies C4A and ApoB in Blood as Potential Markers for Schizophrenia.

The field of proteomics is rapidly gaining territory as a promising alternative to genomic approaches in the efforts to unravel the complex molecular mechanisms underlying schizophrenia and other psychiatric disorders. X-aptamer tech-nology has emerged as a novel proteomic approach for high-sensitivity analyses, and we hypothesized that this technology would identify unique molecular signatures in plasma samples from schizophrenia patients (n = 60) compared to controls (n = 20). Using a combinatorial library of X-aptamer beads, we developed a two-color flow cytometer-based approach to identify specific X-aptamers that bound with high specificity to each target group. Based on this, we synthesized two unique X-aptamer sequences, and specific proteins pulled down from the patient and control groups by these X-aptamers were identified by mass spectrometry. We identified two protein biomarkers, complement component C4A and ApoB, upregulated in plasma samples from schizophrenia patients. ELISA validation suggested that the observed differences in C4 levels in patients are likely due to the presence of the illness itself, while ApoB may be a marker of antipsychotic-induced alterations. These studies highlight the utility of the X-aptamer technology in the identification of biomarkers for schizophrenia that will advance our understanding of the pathophysiological mechanisms of this disorder.

Telomere length in psychiatric disorders: Is it more than an ageing marker?

OBJECTIVES: Psychiatric and substance-use disorders have been associated with premature biological ageing. Telomere length (TL), considered an ageing marker, has been analysed in psychiatric disorders, and to a lesser extent in substance-use disorders, with recent findings suggesting TL may be related to disease pathology. METHODS: We conducted a critical and non-systematic literature search of TL studies published up to June 2016 in psychiatric and substance-use disorders, focussing on studies describing mechanisms, including studies linking telomere biology with genetic factors, stress and mitochondrial alterations (104 studies selected). RESULTS: Patients with major depressive disorder and anxiety appear to have shorter leukocyte telomeres compared to controls. Inconclusive results are found for other psychiatric disorders and for substance-use disorders. This may be due in part to differences in medication treatment and response, as studies suggest that some psychotropic medications may modulate TL. Importantly, some studies establish a relationship between telomere machinery, stress and mitochondria function in psychiatric and substance-use disorders. CONCLUSIONS: While further longitudinal studies considering telomere genetics are needed to clarify the cause-effect link between telomeres and mitochondria function in psychiatric and substance-use disorders, the recent findings linking these biological processes suggest that telomeres may be more than ageing markers.

Anhedonia in cocaine use disorder is associated with inflammatory gene expression.

Treatments for Cocaine Use Disorder (CUD) are variably effective, and there are no FDA-approved medications. One approach to developing new treatments for CUD may be to investigate and target poor prognostic signs. One such sign is anhedonia (i.e. a loss of pleasure or interest in non-drug rewards), which predicts worse outcomes in existing CUD treatments. Inflammation is thought to underlie anhedonia in many other disorders, but the relationship between anhedonia and inflammation has not been investigated in CUD. Therefore, we assessed peripheral genome-wide gene expression in n = 48 individuals with CUD with high (n = 24) vs. low (n = 24) levels of anhedonia, defined by a median split of self-reported anhedonia. Our hypothesis was that individuals with high anhedonia would show differential gene expression in inflammatory pathways. No individual genes were significantly different between the low and high anhedonia groups when using t-tests with a stringent false discovery rate correction (FDR-corrected p < 0.05). However, an exploratory analysis identified 166 loci where t-tests suggested group differences at a nominal p < 0.05. We used DAVID, a bioinformatics tool that provides functional interpretations of complex lists of genes, to examine representation of this gene list in known pathways. It confirmed that mechanisms related to immunity were the top significant associations with anhedonia in the sample. Further, the two top differentially expressed genes in our sample, IRF1 and GBP5, both have primary inflammation and immune functions, and were significantly negatively correlated with total scores on our self-report of anhedonia across all 48 subjects. These results suggest that prioritizing development of anti-inflammatory medications for CUD may pay dividends, particularly in combination with treatment-matching strategies using either phenotypic measures of anhedonia or biomarkers of inflammatory gene expression to individualize treatment.

Disruption of p21-activated kinase 1 gene diminishes atherosclerosis in apolipoprotein E-deficient mice.

Pak1 plays an important role in various cellular processes, including cell motility, polarity, survival and proliferation. To date, its role in atherogenesis has not been explored. Here we report the effect of Pak1 on atherogenesis using atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice as a model. Disruption of Pak1 in ApoE(-/-) mice results in reduced plaque burden, significantly attenuates circulating IL-6 and MCP-1 levels, limits the expression of adhesion molecules and diminishes the macrophage content in the aortic root of ApoE(-/-) mice. We also observed reduced oxidized LDL uptake and increased cholesterol efflux by macrophages and smooth muscle cells of ApoE(-/-):Pak1(-/-) mice as compared with ApoE(-/-) mice. In addition, we detect increased Pak1 phosphorylation in human atherosclerotic arteries, suggesting its role in human atherogenesis. Altogether, these results identify Pak1 as an important factor in the initiation and progression of atherogenesis.

Metanx alleviates multiple manifestations of peripheral neuropathy and increases intraepidermal nerve fiber density in Zucker diabetic fatty rats.

Metanx is a product containing L-methylfolate, pyridoxal 5'-phosphate, and methylcobalamin for management of endothelial dysfunction. Metanx ingredients counteract endothelial nitric oxide synthase uncoupling and oxidative stress in vascular endothelium and peripheral nerve. This study evaluates Metanx on diabetic peripheral neuropathy in ZDF rats, a model of type 2 diabetes. Metanx was administered to 15-week-old ZDF and ZDF lean rats at either 4.87 mg ⋅ kg(-1) ⋅ day(-1) (a body weight-based equivalent of human dose) or 24.35 mg ⋅ kg(-1) ⋅ day(-1) by oral gavage two times a day for 4 weeks. Both doses alleviated hind limb digital sensory, but not sciatic motor, nerve conduction slowing and thermal and mechanical hypoalgesia in the absence of any reduction of hyperglycemia. Low-dose Metanx increased intraepidermal nerve fiber density but did not prevent morphometric changes in distal tibial nerve myelinated fibers. Metanx treatment counteracted endothelial nitric oxide synthase uncoupling, inducible nitric oxide synthase upregulation, and methylglyoxal-derived advanced glycation end product, nitrotyrosine, and nitrite/nitrate accumulation in the peripheral nerve. In conclusion, Metanx, at a body weight-based equivalent of human dose, increased intraepidermal nerve fiber density and improved multiple parameters of peripheral nerve function in ZDF rats. Clinical studies are needed to determine if Metanx finds use in management of diabetic peripheral neuropathy.