25-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages

Recent studies have shown that Toll-like receptor (TLR)- signalling contributes significantly to the inflammatory events of atherosclerosis. As products of cholesterol oxidation (oxysterols) accumulate within atherosclerotic plaque and have been proposed to contribute to inflammatory signalling in the diseased artery, we investigated the potential of 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-HC) and 25-hydroxycholesterol (25-HC) to stimulate inflammatory signalling via the lipid-recognising TLRs 1, 2, 4 and 6. Each oxysterol stimulated secretion of the inflammatory chemokine interleukin-8 (IL-8), but not I?B degradation or tumour necrosis factor- release from monocytic THP-1 cells. Transfection of TLR-deficient HEK-293 cells with TLRs 1, 2, 4 or 6 did not increase sensitivity to the tested oxysterols. Moreover, blockade of TLR2 or TLR4 with specific inhibitors did not reduce 25-hydroxycholesterol (25-HC) induced IL-8 release from THP-1 cells. We conclude that although the oxysterols examined in this study may contribute to increased expression of certain inflammatory genes, this occurs by mechanisms independent of TLR signalling.

Can the biology of VEGF and haem oxygenases help solve pre-eclampsia?

Pre-eclampsia, a pregnancy-specific multi-organ syndrome characterized by widespread endothelial damage, is a new risk factor for cardiovascular disease. No therapies exist to prevent or treat this condition, even to achieve a modest improvement in pregnancy length or birth weight. Co-administration of soluble VEGFR-1 [VEGF (vascular endothelial growth factor) receptor-1; more commonly known as sFlt-1 (soluble Fms-like tyrosine kinase-1)] and sEng (soluble endoglin) to pregnant rats elicits severe pre-eclampsia-like symptoms. These two anti-angiogenic factors are increased dramatically prior to the clinical onset of pre-eclampsia and are quite possibly the 'final common pathway' responsible for the accompanying signs of hypertension and proteinuria as they can be reversed by VEGF administration in animal models. HO-1 (haem oxygenase-1), an anti-inflammatory enzyme, and its metabolite, CO (carbon monoxide), exert protective effects in several organs against oxidative stimuli. In a landmark publication, we showed that the HO-1 pathway inhibits sFlt-1 and sEng in cultured cells and human placental tissue explants. Both CO and NO (nitric oxide) promote vascular homoeostasis and vasodilatation, and activation of VEGFR-1 or VEGFR-2 induced eNOS (endothelial nitric oxide synthase) phosphorylation, NO release and HO-1 expression. Our studies established the HO-1/CO pathway as a negative regulator of cytokine-induced sFlt-1 and sEng release and eNOS as a positive regulator of VEGF-mediated vascular morphogenesis. These findings provide compelling evidence for a protective role of HO-1 in pregnancy and identify it as a target for the treatment of pre-eclampsia. Any agent that is known to up-regulate HO-1, such as statins, may have potential as a therapy. Any intervention achieving even a modest prolongation of pregnancy or amelioration of the condition could have a significant beneficial health impact worldwide.

Modulation of glucagon receptor pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)

The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, whilst having clinical efficacy, have been associated with severe adverse side-effects and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems, to provide a more complete understanding of glucagon receptor signaling considering the effect of multiple ligands, association with the receptor-interacting protein, receptor activity modifying protein-2 (RAMP2) and individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.

A novel role of plasma membrane calcium atpase 4 as a negative-regulator of VEGF-induced angiogenesis

Current anti-angiogenic treatments involve the attenuation of signalling via the pro-angiogenic vascular endothelial growth factor/receptor (VEGF/VEGFR) axis. Stimulation of angiogenesis by VEGF requires the activation of the calcineurin/nuclear factor of activated T-cells (NFAT) signal transduction pathway which is inhibited by Plasma Membrane Calcium ATPase 4 (PMCA4), an endogenous calcium extrusion pump. However, PMCA4s role in calcineurin/NFAT-dependent angiogenesis is unknown. Using “gain of function” studies, we show here that adenoviral overexpression of PMCA4 in human umbilical vein endothelial cells (HUVEC) inhibited NFAT activity, decreased the expression of NFAT-dependent pro-angiogenic proteins (regulator of calcineurin 1.4 (RCAN1.4) and cyclooxygenase-2) and diminished in vitro cell migration and tube formation in response to VEGF-stimulation. Furthermore, in vivo blood vessel formation was attenuated in a matrigel plug assay by ectopic expression of PMCA4. Conversely, “loss of function” experiments by si-RNA-mediated knockdown of PMCA4 in HUVEC or isolation of mouse lung endothelial cells from PMCA4−/− mice showed increased VEGF-induced NFAT activity, RCAN1.4 expression, in vitro endothelial cell migration, tube formation and in vivo blood vessel formation. Additionally, in an in vivo pathological angiogenesis model of limb ischemia, the reperfusion of the ischemic limb of PMCA4−/− mice was augmented compared to wild-type. Disruption of the interaction between endogenous PMCA4 and calcineurin by adenoviral overexpression of the region of PMCA4 that interacts with calcineurin (residues 428–651) increased NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify PMCA4 as an inhibitor of VEGF-induced angiogenesis, highlighting its potential as a new therapeutic target for anti-angiogenic treatments.

Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

Cholecystokinin-1 receptor antagonists:5-hydroxy-5-aryl-pyrrol-2-ones as anticancer agents

A new class of 5-arylated 5-hydroxypyrrolones was derived from mucochloric acid in 2 synthetic steps and the chemical structure was confirmed additionally by X-ray analysis. Using a radiolabelled binding assay, potent CCK1 selective ligands were identified (CCK1: 12 nM) and the antagonism was confirmed by using isolated tissue preparations. A series of isobutyl derivatives displayed unsurmountable CCK antagonistic properties and in vitro excellent inhibition of proliferation was obtained in cholecystokinin related cancer cell lines in the nanomolar range. Finally, using xenograft studies in nude mice, two selected pyrrolone derivatives, X = H and X = F a fluorinated analogue (PNB-028), showed a strong inhibition of tumour growth in a chemo-resistant colon cancer-(MAC 16) and a human pancreatic cell line (MIAPACA) at 50 mg kg-1 by oral administration.