Mette M. Rosenkilde

The chemokine system – a major regulator of angiogenesis in health and disease

The chemokine system controls leukocyte trafficking during homeostasis as well as during inflammation and is necessary for the linkage between innate and adaptive immunity. Tissue regulation outside the hematopoietic compartment, for instance, angiogenesis, organogenesis and tumor development, growth and metastasis, is another important function of the chemokine system. The chemokine‐mediated regulation of angiogenesis is highly sophisticated and fine tuned, and involves pro‐angiogenic chemokines, for instance, CXCL8/IL8 interacting with the CXCR2 receptor, and anti‐angiogenic (i.e. angiostatic) chemokines, for instance, CXCL10/IP10 interacting with the CXCR3 receptor. Chemokines also regulate angiogenesis in a receptor‐independent manner by means of a perturbation of bFGF and VEGF function. The current review focuses on the influence of the chemokine system in angiogenesis. Examples of the delicate angiogenesis regulation by the chemokine system in, for instance, wound healing and of the dysregulation in, for instance, tumor development are provided along with the interesting phenomenon of molecular piracy of host‐encoded genes within the chemokine system. This phenomenon is a general strategy to circumvent and exploit the immune system – and thereby improve survival – for many viruses. Yet, a certain group of herpesviruses – the γ2‐herpesviruses – encode a functional CXCR2 receptor homolog that is activated by angiogenic chemokines and antagonized by angiostatic chemokines, and this particular gene seems to cause the development of a vascular tumor – Kaposis sarcoma – in the host.

Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28

The 7TM receptor, US28, encoded by human cytomegalovirus binds a broad spectrum of endogenous CC chemokines with sub‐nanomolar affinity as determined in homologous competition binding assays. We here find that US28 also recognizes the membrane‐associated CX3C chemokine, fractalkine, with sub‐nanomolar affinity (IC50=0.42±0.09 nM). Importantly, although fractalkine could compete with high affinity against the binding of CC chemokines, the secreted CC chemokines were only able to compete for binding against radioactive fractalkine with very low affinity. It is concluded that US28, which is known to enhance cell‐cell fusion processes through interaction with an as yet unidentified, human cell‐specific factor, has been optimized by cytomegalovirus to selectively recognize the membrane‐associated fractalkine. It is suggested that US28 expressed on the surface of infected cells and possibly on the envelope of the virion is involved in transfer of the virus from cell to cell.

2-Oleoyl Glycerol Is a GPR119 Agonist and Signals GLP-1 Release in Humans

OBJECTIVEnDietary fat is thought to stimulate release of incretin hormones via activation of fatty acid receptors in the intestine. However, dietary fat (triacylglycerol) is digested to 2-monoacylglycerol and fatty acids. Activation of G protein-coupled receptor 119 (GPR119) stimulates glucagon-like peptide-1 (GLP-1) release from the intestinal L-cells. We aimed to investigate if 2-oleoyl glycerol (2OG) can activate GPR119 in vitro and stimulate GLP-1 secretion in vivo.nnnRESEARCH DESIGN AND METHODSnAgonist activity for various lipids was tested on transiently expressed human GPR119 in COS-7 cells. The effect of a jejunal bolus of 2 g 2OG on plasma levels of GLP-1 was evaluated in eight healthy human volunteers. The effect of 2OG was compared to an equimolar amount of oleic acid, a degradation product from 2OG, and the vehicle, glycerol. Digestion of 5 ml olive oil with pancreatic lipase will result in formation of approximately 2 g 2OG and 3.2 g oleic acid.nnnRESULTSn2OG and other 2-monoacylglycerols increased intracellular concentrations of cAMP in GPR119-expressing COS-7 cells (2OG EC(50) = 2.5 μm). Administration of 2OG to humans significantly increased plasma GLP-1 (0-25 min) when compared to the two controls, oleic acid and vehicle. Plasma levels of glucose-dependent insulinotropic polypeptide also increased.nnnCONCLUSIONn2OG and other 2-monoacylglycerols formed during fat digestion can activate GPR119 and cause incretin release from the human intestine. This mechanism is likely to contribute to the known stimulatory effect of dietary fat on incretin secretion, and it indicates that GPR119 is a fat sensor.

Natural agonist enhancing bis-His zinc-site in transmembrane segment V of the tachykinin NK3 receptor

In the wild‐type tachykinin NK3A receptor histidyl residues are present at two positions in TM‐V, V:01 and V:05, at which Zn2+ functions as an antagonist in NK1 and κ‐opioid receptors with engineered metal‐ion sites. Surprisingly, in the NK3A receptor Zn2+ instead increased the binding of the agonist 125I‐[MePhe7]neurokinin B to 150%. [MePhe7]neurokinin B bound to the NK3A receptor in a two‐component mode of which Zn2+ eliminated the subnanomolar binding mode but induced a higher binding capacity of the nanomolar binding mode. Signal transduction was not induced by ZnCl2 but 10 μM ZnCl2 enhanced the effect of neurokinin B. Ala‐substitution of HisV:01 eliminated the enhancing effect of Zn2+ on peptide binding. It is concluded that physiological concentrations of Zn2+ have a positive modulatory effect on the binding and function of neurokinin B on the NK3A receptor through a bis‐His site in TM‐V.

Mutations at the CXCR4 interaction sites for AMD3100 influence anti-CXCR4 antibody binding and HIV-1 entry

The interaction of the CXCR4 antagonist AMD3100 with its target is greatly influenced by specific aspartate residues in the receptor protein, including Asp171 and Asp262. We have now found that aspartate‐to‐asparagine substitutions at these positions differentially affect the binding of four different anti‐CXCR4 monoclonal antibodies as well as the infectivity of diverse human immunodeficiency virus type 1 (HIV‐1) strains and clinical isolates. Mutation of Asp262 strongly decreased the coreceptor efficiency of CXCR4 for wild‐type but not for AMD3100‐resistant HIV‐1 NL4.3. Thus, resistance of HIV‐1 NL4.3 to AMD3100 is associated with a decreased dependence of the viral gp120 on Asp262 of CXCR4, pointing to a different mode of interaction of wild‐type versus AMD3100‐resistant virus with CXCR4.

Targeting CXCR4 in HIV cell-entry inhibition.

CXCR4 and CCR5 constitute the two major coreceptors for HIV-1 entry into host cells. In the course of an HIV-infection, a coreceptor switch takes place in approximately half of the patients - from R5 HIV-1 (CCR5 utilizing) strains to X4 HIV-1 (CXCR4 utilizing) strains. Treatment of HIV-infected individuals with CXCR4 antagonists delays the onset of AIDS by preventing the CCR5 to CXCR4 coreceptor switch. In addition to the endogenous CXCR4 and CCR5 ligands, other chemokines, for example the human herpesvirus 8 encoded CC-chemokine, vCCL2, and modifications hereof, have proven efficient HIV-1 cell-entry inhibition through chemokine receptor interaction. However, pharmacokinetic and immunogenic drawbacks of chemokines and peptidic/peptoid compounds have brought the attention towards small-molecule antagonists, such as AMD3100, that displays high specificity and affinity towards CXCR4, but unfortunately no oral bioavailability. The hunt for orally active small-molecule CXCR4 antagonists led to the development of monocyclam-based compounds, and recently to the non-cyclam antagonist AMD070, which is orally active and currently in Phase II clinical trial as anti-HIV treatment. Current review provides an overview of the drug discovery within the field of anti-HIV treatment targeting CXCR4 spanning from natural occurring and modified chemokines, to HIV-mimicking peptides and peptoids ending at the non-peptide antagonists.

Virally encoded chemokines and chemokine receptors in the role of viral infections.

Large DNA viruses such as pox- and in particular herpesviruses are notorious in their ability to evade the immune system and to be maintained in the general population. Based on the accumulated knowledge reviewed in this study it is evident that important mechanisms of these actions are the acquisition and modification of host-encoded chemokines and chemokine receptors. The described viral molecules leave nothing to chance and have thoroughly and efficiently corrupted the host immune system. Through this process viruses have identified key molecules in antiviral responses by their inhibition of these or potent ways to alter an efficient antiviral response to a weak Th2-driven response. Examples here are the chemokine scavenging by US28, attractance of Th2 cells and regulatory cells by vMIP1-3 and the selective engaging of CCR8 by MC148. Important insights into viral pathology and possible targets for antiviral therapies have been provided by UL33, UL78 and in particular ORF74 and the chances are that many more will follow. In HHV8 vMIP-2 and the chemokine-binding proteins potent anti-inflammatory agents have been provided. These have already had their potential demonstrated in animal models and may in their native or modified forms represent useful therapies in humans.