Perivascular Expression and Potent Vasoconstrictor Effect of Dynorphin A in Cerebral Arteries

BACKGROUND: Numerous literary data indicate that dynorphin A (DYN-A) has a significant impact on cerebral circulation, especially under pathophysiological conditions, but its potential direct influence on the tone of cerebral vessels is obscure. The aim of the present study was threefold: 1) to clarify if DYN-A is present in cerebral vessels, 2) to determine if it exerts any direct effect on cerebrovascular tone, and if so, 3) to analyze the role of κ-opiate receptors in mediating the effect. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analysis revealed the expression of DYN-A in perivascular nerves of rat pial arteries as well as in both rat and human intraparenchymal vessels of the cerebral cortex. In isolated rat basilar and middle cerebral arteries (BAs and MCAs) DYN-A (1-13) and DYN-A (1-17) but not DYN-A (1-8) or dynorphin B (DYN-B) induced strong vasoconstriction in micromolar concentrations. The maximal effects, compared to a reference contraction induced by 124 mM K(+), were 115±6% and 104±10% in BAs and 113±3% and 125±9% in MCAs for 10 µM of DYN-A (1-13) and DYN-A (1-17), respectively. The vasoconstrictor effects of DYN-A (1-13) could be inhibited but not abolished by both the κ-opiate receptor antagonist nor-Binaltorphimine dihydrochloride (NORBI) and blockade of G(i/o)-protein mediated signaling by pertussis toxin. Finally, des-Tyr(1) DYN-A (2-13), which reportedly fails to activate κ-opiate receptors, induced vasoconstriction of 45±11% in BAs and 50±5% in MCAs at 10 µM, which effects were resistant to NORBI. CONCLUSION/SIGNIFICANCE: DYN-A is present in rat and human cerebral perivascular nerves and induces sustained contraction of rat cerebral arteries. This vasoconstrictor effect is only partly mediated by κ-opiate receptors and heterotrimeric G(i/o)-proteins. To our knowledge our present findings are the first to indicate that DYN-A has a direct cerebral vasoconstrictor effect and that a dynorphin-induced vascular action may be, at least in part, independent of κ-opiate receptors

Reactive oxygen species mediate a cellular 'memory' of high glucose stress signalling

Aims/hypothesis A long-term 'memory' of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding. Materials and methods HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus alpha-lipoic acid in the last week. Results In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-beta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADP-ribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant alpha-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H(2)DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with alpha-lipoic acid added to the last week of normalised glucose. Conclusions/interpretation These results provide proof-of-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation

Iron Oxide Nanoparticle-Mediated Development of Cellular Gap Junction Crosstalk to Improve Mesenchymal Stem Cells’ Therapeutic Efficacy for Myocardial Infarction

Electrophysiological phenotype development and paracrine action of mesenchymal stem cells (MSCs) are the critical factors that determine the therapeutic efficacy of MSCs for myocardial infarction (MI). In such respect, coculture of MSCs with cardiac cells has windowed a platform for cardiac priming of MSCs. Particularly, active gap junctional crosstalk of MSCs with cardiac cells in coculture has been known to play a major role in the MSC modification through coculture. Here, we report that iron oxide nanoparticles (IONPs) significantly augment the expression of connexin 43 (Cx43), a gap junction protein, of cardiomyoblasts (H9C2), which would be critical for gap junctional communication with MSCs in coculture for the generation of therapeutic potential-improved MSCs. MSCs cocultured with IONP-harboring H9C2 (cocultured MSCs: cMSCs) showed active cellular crosstalk with H9C2 and displayed significantly higher levels of electrophysiological cardiac biomarkers and a cardiac repair-favorable paracrine profile, both of which are responsible for MI repair. Accordingly, significantly improved animal survival and heart function were observed upon cMSC injection into rat MI models compared with the injection of unmodified MSCs. The present study highlights an application of IONPs in developing gap junctional crosstalk among the cells and generating cMSCs that exceeds the reparative potentials of conventional MSCs. On the basis of our finding, the potential application of IONPs can be extended in cell biology and stem cell-based therapies