Fibrinolysis inhibitors in plaque stability: a morphological association of PAI-1 and TAFI in advanced carotid plaque.

Essentials Fibrinolysis inhibitors are localized in advanced atheroma by immunohistology of endarterectomies. Neovascular endothelium/neocapillaries show thrombin-activatable fibrinolysis inhibitor (TAFI). Macrophage areas show free plasminogen activator inhibitor (PAI-1), notably in the vulnerable part. Free PAI-1 and TAFI stabilize active plaque area by inhibition of fibrinolysis and inflammation. SUMMARY: Background Fibrinolysis plays an important role in destabilization of atherosclerotic plaques and is tightly regulated by specific inhibitors. Objective The fibrinolysis inhibitors plasminogen activator inhibitor type-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were quantified and described in the morphological context of advanced carotid plaques American Heart Association VI-VIII to elucidate their role in plaque stability. Methods Immunohistochemistry in serial sections along the longitudinal axis of endarterectomies from patients with symptomatic carotid stenosis (n = 19) were studied using an antibody specific for free PAI-1 (I205), an antibody with high affinity for TAFI/TAFIa (CP17) and established antibodies for smooth muscle cells (α-actin), endothelial cells (von Willebrand factor [VWF]), macrophages (CD68) and platelets (CD42). Results PAI-1 and TAFI show a specific distribution in these advanced plaques with a maximum corresponding to the internal carotid artery (ICA). Free PAI-1 was mainly detected in macrophages and in intravascular thrombi, and TAFI in endothelial cells (ECs) but also macrophages. The one-way ANOVA analysis with Bonferroni's correction showed a significant increase of macrophages and ECs, TAFI and PAI-1 in areas with high neovascularization in endarterectomy sections corresponding to ICA. High Spearman factors for TAFI, PAI-1 and VWF indicate neovascularization as the main source of plasma proteins, transported by platelets into the atheroma (PAI-1) or expressed by ECs (TAFI). CD68 was highly associated with VWF, PAI-1 and especially TAFI, underlining the role of macrophages in fibrinolytic activity and inflammation. Conclusion The abundance of free PAI-1 and TAFI in the plaque may inhibit plasmin generation and thereby counteract plaque destabilization by fibrinolysis, cell migration and inflammation.

Neuropeptide Y and its Y2 receptor: potential targets in neuroblastoma therapy.

Neuroblastomas are pediatric tumors that develop from sympathetic precursors and express neuronal proteins, such as neuropeptide Y (NPY). NPY is a sympathetic neurotransmitter acting via multiple receptors (Y1-Y5R). Both NPY and Y2Rs are commonly expressed in neuroblastoma cell lines and tissues. The peptide secreted from neuroblastomas stimulates tumor cell proliferation and angiogenesis. As both processes are Y2R-mediated, the aim of this study was to assess Y2R as a potential therapeutic target for neuroblastoma. In vitro, Y2R antagonist (BIIE0246) prevented activation of p44/42 mitogen-activated protein kinase (MAPK) induced by endogenous NPY, which resulted in decreased proliferation and induction of Bim-mediated apoptosis. Similar growth-inhibitory effects were achieved with NPY small interfering RNA (siRNA) and Y2R siRNA. In vivo, Y2R antagonist significantly inhibited growth of SK-N-BE(2) and SK-N-AS xenografts, which was associated with decreased activation of p44/42 MAPK, as well as reduced proliferation (Ki67) and increased apoptosis (TdT-mediated dUTP nick end labeling; TUNEL). The Y2R antagonist also exerted an antiangiogenic effect. In vitro, it reduced the proliferation of endothelial cells induced by neuroblastoma-conditioned media. Consequently, the Y2R antagonist-treated xenografts had decreased vascularization and a high degree of focal fibrosis. In human neuroblastoma tissues, the expression of Y2R was observed in both tumor and endothelial cells, while NPY was predominantly expressed in neuroblastoma cells. In summary, Y2R is a promising new target for neuroblastoma therapy affecting both cancer cells and tumor vasculature.

The role of GABA(A) receptors in the control of transient lower oesophageal sphincter relaxations in the dog.

BACKGROUND AND PURPOSE: Transient lower oesophageal sphincter relaxations (TLESRs) are triggered by activation of mechanosensitive gastric vagal afferents and are the major cause of gastroesophageal reflux and therefore an important target for therapeutic intervention in gastroesophageal reflux disease (GERD). Activation of the metabotropic GABA(B) receptor has shown to inhibit TLESRs. The aim of the present study was to assess the role of the ionotropic GABA(A) receptor in the regulation of TLESRs. EXPERIMENTAL APPROACH: TLESRs were quantified using Dentsleeve manometry in dogs, and GABA(A) agonists were given i.v. prior to gastric distension. Immunohistochemistry and RT-PCR were used to localize GABA(A) receptors in the dog nodose ganglion, the source of vagal afferents which initiate TLESRs. KEY RESULTS: The prototypical GABA(A) agonist muscimol produced a dose-dependent inhibition of TLESRs ranging from 19 to 56%. The two other GABA(A) agonists evaluated, isoguvacine and 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), as well as the GABA(A) positive allosteric modulator diazepam, had no major effects on TLESRs. Evaluation of higher doses was limited by emesis (THIP and isoguvacine) or restlessness/sedation (diazepam). Of the predominant GABA(A) receptor subunits (alpha, beta and gamma components), alpha and beta but not gamma were detected in the dog nodose ganglion by RT-PCR, while immunohistochemistry in addition demonstrated nerve fibres expressing the gamma subunit. CONCLUSIONS AND IMPLICATIONS: The present observations demonstrate that GABA(A) receptors exert an inhibitory control of TLESRs. These results warrant further studies using GABA(A) isoform-selective agonists to define the identity of receptors involved.

Characterization of the cloned atlantic cod neuropeptide Y-Yb receptor: peptide-binding requirements distinct from known mammalian Y receptors.

Five members of the neuropeptide Y (NPY) receptor family have been cloned in mammals. The recently cloned NPY receptor in the Atlantic cod seems to be distinct from the mammalian subtypes as it has only 50% identity to Y1, Y4, and y6 and only 30% to Y2 and Y5. In most of the other families of G-protein-coupled receptors, species homologues have 65-90% identity between fishes and mammals. The functional expression and detailed pharmacological characterization of this cod NPY receptor, designated Yb, is reported. Membranes of cells transiently transfected with cod Yb showed saturable [(125)I]PYY binding with a K(d) of 45 pM. The pharmacological profile is similar to those of both the zebrafish Yb and Yc receptors and distinct from those of the mammalian NPY receptors. In competition experiments the cod Yb receptor had the following rank order of potencies: porcine PYY = porcine NPY = p[Leu(31), Pro(34)]NPY > zebrafish PYY > zebrafish NPY >> NPY2-36 = NPY3-36 > NPY18-36 > bovine PP = [D-Trp(32)]NPY > BIBP3226. This is in sharp contrast to the high selectivity of BIBP3226 for the Y1 receptor from all mammalian species. Together with the low amino acid identity of cod Yb with the mammalian Y1, Y4, and y6 receptors, this is further support for the notion that fish Yb constitutes a distinct NPY receptor subtype.

Cloning of a neuropeptide Y/peptide YY receptor from the Atlantic cod: the Yb receptor.

Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides that bind to G protein-coupled receptors. Five different receptor subtypes have recently been cloned in mammals and we have found another three receptor genes in the zebrafish, called zYa, zYb, and zYc, that appear to be distinct subtypes as deduced from their widely different sequences. To elucidate the evolutionary relationships between the mammalian and zebrafish receptors, we have used the zebrafish probes to isolate genomic clones from another teleost fish, the Atlantic cod, Gadus morhua. We present here the sequence of the cod Yb gene, whose deduced protein sequence is equally identical to the zebrafish Yb (69%) and Yc proteins (66%). The two zebrafish receptors are 76% identical to each other, suggesting that they arose by gene duplication in the zebrafish lineage after divergence from the cod lineage. The five cloned mammalian NPY-family receptors and the three cloned zebrafish NPY receptors indicate that this is the largest receptor family among all peptide receptors that belong to the superfamily of G protein-coupled receptors.