Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins

The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC-chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC-ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC-ClOH.     

Increase in the growth inhibition of bovine pulmonary artery smooth muscle cells by an O-hexanoyl low-molecular-weight heparin derivative

Proliferation of pulmonary artery smooth muscle cells (PASMCs) appears to play a significant role in chronic pulmonary hypertension. The proliferation of PASMCs is strongly inhibited by some commercial heparin preparations. Heparin fragments were prepared by periodate treatment, followed by sodium borohydride reduction, to enhance potency. The tributylammonium salt of this fragmented heparin was O-acylated with hexanoic anhydride. Gradient polyacrylamide gel electrophoresis showed that the major heparin fragment contained eight disaccharide units. NMR analysis showed that approximately one hexanoyl group per disaccharide residue was present. The O-hexanoyl heparin fragments were assayed for growth inhibitory effect on bovine PASMCs in culture. This derivative was found to be more effective in growth inhibition of bovine PASMCs in culture than the heparin from which it was derived. In the future, it is envisioned that this or similar derivatives may be an effective treatment for pulmonary hypertension.     

CpG-enriched plasmid enhances the efficacy of the traditional foot-and-mouth disease killed vaccine

A CpG-enriched recombinant plasmid (pUC18-CpG) as an adjuvant of FMD killed vaccine was tested for immunization and vaccination challenge in a porcine model. Our preliminary results had indicated that the recombinant plasmid could enhance the humoral immune response triggered by the traditional oil-adjuvant vaccine after the initial inoculation. A subsequent vaccination-challenge test showed an increased PD(50) value. Thus, coadministration of the recombinant plasmid with the oil-adjuvant vaccine helped illicit an immune response earlier than that elicited by giving the vaccine alone. Our results showed that pUC18-CpG can be a potent immunoadjuvant for the traditional FMD killed vaccine and can greatly enhance the traditional vaccine's efficacy when given in combination with it.     

Identification of differentially expressed microRNAs and their PKC-isoform specific gene network prediction during hypoxic pre-conditioning and focal cerebral ischemia of mice

We previously reported the involvement of conventional protein kinase C (cPKC) βII, γ, novel PKC (nPKC) ε and their interacting proteins in hypoxic pre-conditioning (HPC)-induced neuroprotection. In this study, the large-scale miRNA microarrays and bioinformatics analysis were used to determine the differentially expressed miRNAs and their PKC-isoform specific gene network in mouse brain after HPC and 6?h middle cerebral artery occlusion (MCAO). We found 4 up-regulated and 13 down-regulated miRNAs in the cortex of HPC mice, 26 increased and 39 decreased gene expressions of miRNAs in the peri-infarct region of 6?h MCAO mice, and 11 up-regulated and 22 down-regulated miRNAs in the peri-infarct region of HPC and 6?h MCAO mice. Based on Diff Score, 19 differentially expressed miRNAs were identified in HPC and 6?h MCAO mouse brain. Then the miRNA-gene-network of 19 specified miRNAs target genes of cPKCβII, γ and nPKCε-interacting protein was predicted by using bioinformatics analysis of genome databases. Furthermore, the down-regulated miR-615-3p during HPC had a detrimental effect on the oxygen-glucose deprivation (OGD)-induced N2A cell injury. These results suggested that the identified 19 miRNAs, notably miR-615-3p, might target these genes of cPKCβII, γ and nPKCε-interacting proteins involved in HPC-induced neuroprotection.     

The bioactive peptides salusins and apelin-36 are produced in human arterial and venous tissues and the changes of their levels during cardiopulmonary bypass

This study aimed to examine the effects of CPB on salusin-α, salusin-β and apelin-36 bioactive peptides in people who are planned to undergo coronary artery bypass graft (CABG) operation due to coronary artery disease and to explore whether these peptides are produced in human aortic, saphenous and arterial tissues. The study included age and BMI matched 15 patients who underwent CABG operation by CPB. In order to determine salusin-α, salusin-β and apelin-36 levels, venous blood samples were collected before induction of anesthesia (T1), before CPB (T2), 5min before the removal of cross-clamp (T3), 5min after the removal of cross-clamp (T4), upon arrival in the intensive care (T5), at postoperative 24th hour (T6) and 72nd hour (T7). Salusin and apelin expressions of the tissues were shown by immunohistochemical method. Peptide amounts of sera and tissues were measured using ELISA. Salusins production by vessels occurs in fibroblast cells of the media in the aorta and smooth muscle cells of the media in the LIMA and saphena. Apelin is produced by endothelial cells of the intima and fibroblast cells of the media in the aorta and by smooth muscle cells of the media in the LIMA and saphena. Changes in the levels of salusin-β and apelin-36 were significant during CPB. Salusin-α, salusin-β and apelin-36 are locally synthesized in the arteries and veins. Salusins and apelin-36 might be important markers in the CPB, and also that salusin-β was more specific in comparison to salusin-α.     

A derivative of the CRMP2 binding compound lanthionine ketimine provides neuroprotection in a mouse model of cerebral ischemia

Lanthionines are novel neurotrophic and neuroprotective small molecules that show promise for the treatment of neurodegenerative diseases. In particular, a recently developed, cell permeable lanthionine derivative known as LKE (lanthionine ketimine 5-ethyl ester) promotes neurite growth at low nanomolar concentrations. LKE also has neuroprotective, anti-apoptotic, and anti-inflammatory properties. Its therapeutic potential in cerebral ischemia and its mechanisms of neurotrophic action remain to be fully elucidated. Here, we hypothesize that the neuroprotective actions of LKE could result from induction or modulation of CRMP2. We found that treating primary cultured mouse neurons with LKE provided significant protection against t-butyl hydroperoxide-induced neuronal death possibly through CRMP2 upregulation. Similarly, in vivo studies showed that LKE pre and/or post-treatment protects mice against permanent distal middle cerebral artery occlusion (p-MCAO) as evidenced by lower stroke lesions and improved functional outcomes in terms of rotarod, grip strength and neurologic deficit scores in treated groups. Protein expression levels of CRMP2 were higher in brain cortices of LKE pretreated mice, suggesting that LKE’s neuroprotective activity may be CRMP2 dependent. Lower activity of cleaved PARP-1 and higher activity of SIRT-1 was also observed in LKE treated group suggesting its anti-apoptotic properties. Our results suggest that LKE has potential as a therapeutic intervention in cerebral ischemia and that part of its protective mechanism may be attributed to CRMP2 mediated action and PARP-1/SIRT-1 modulation.     

Conditionally immortalised neural stem cells promote functional recovery and brain plasticity after transient focal cerebral ischaemia in mice

Cell therapy has enormous potential to restore neurological function after stroke. The present study investigated effects of conditionally immortalised neural stem cells (ciNSCs), the Maudsley hippocampal murine neural stem cell line clone 36 (MHP36), on sensorimotor and histological outcome in mice subjected to transient middle cerebral artery occlusion (MCAO).Adult male C57BL/6 mice underwent MCAO by intraluminal thread or sham surgery and MHP36 cells or vehicle were implanted into ipsilateral cortex and caudate 2 days later. Functional recovery was assessed for 28 days using cylinder and ladder rung tests and tissue analysed for plasticity, differentiation and infarct size.MHP36-implanted animals showed accelerated and augmented functional recovery and an increase in neurons (MAP-2), synaptic plasticity (synaptophysin) and axonal projections (GAP-43) but no difference in astrocytes (GFAP), oligodendrocytes (CNPase), microglia (IBA-1) or lesion volumes when compared to vehicle group.This is the first study showing a potential functional benefit of the ciNSCs, MHP36, after focal MCAO in mice, which is probably mediated by promoting neuronal differentiation, synaptic plasticity and axonal projections and opens up opportunities for future exploitation of genetically altered mice for dissection of mechanisms of stem cell based therapy.     

Secretome analysis of atherosclerotic and non-atherosclerotic arteries reveals dynamic extracellular remodeling during pathogenesis

Aims

Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue secretome and to the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development.

Methods and results

Secretomes from three biological replicates of human atherosclerotic coronary arteries (APC), preatherosclerotic coronaries (PC) and mammaries (M) were analyzed by LC-MS/MS. The identified proteins were submitted to Ingenuity Pathway Analysis (IPA) tool. Label-free MS/MS based quantification was performed and validated by immunohistochemistry. 64 proteins were identified in the 3 replicates of at least one of the 3 groups and 15 secreted proteins have not been previously reported in plasma. Four proteins were significantly released in higher amounts by mammary tissue: gelsolin, vinculin, lamin A/C and phosphoglucomutase 5.

Conclusion

The study of tissue secretome reveals key proteins involved in atherosclerosis which have not been previously reported in plasma. Novel proteins are here highlighted which could be potential therapeutic targets in clinical practice. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.