An IP-10 (CXCL10)-derived peptide inhibits angiogenesis

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77-98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis. © 2012 Yates-Binder et al

Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines

yesWe describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokine receptors migrate against this gradient by crawling under the agarose spots towards their centre. We show that this migration is chemokine-specific; meaning that only cells that express the cognate chemokine cell surface receptor, migrate under the spot containing its corresponding chemokine ligand. Furthermore, we show that migration under the agarose spot can be modulated by selective small molecule antagonists present in the cell culture medium

Protein Translation and Cell Death: The Role of Rare tRNAs in Biofilm Formation and in Activating Dormant Phage Killer Genes

We discovered previously that the small Escherichia coli proteins Hha (hemolysin expression modulating protein) and the adjacent, poorly-characterized YbaJ are important for biofilm formation; however, their roles have been nebulous. Biofilms are intricate communities in which cell signaling often converts single cells into primitive tissues. Here we show that Hha decreases biofilm formation dramatically by repressing the transcription of rare codon tRNAs which serves to inhibit fimbriae production and by repressing to some extent transcription of fimbrial genes fimA and ihfA. In vivo binding studies show Hha binds to the rare codon tRNAs argU, ileX, ileY, and proL and to two prophage clusters D1P12 and CP4-57. Real-time PCR corroborated that Hha represses argU and proL, and Hha type I fimbriae repression is abolished by the addition of extra copies of argU, ileY, and proL. The repression of transcription of rare codon tRNAs by Hha also leads to cell lysis and biofilm dispersal due to activation of prophage lytic genes rzpD, yfjZ, appY, and alpA and due to induction of ClpP/ClpX proteases which activate toxins by degrading antitoxins. YbaJ serves to mediate the toxicity of Hha. Hence, we have identified that a single protein (Hha) can control biofilm formation by limiting fimbriae production as well as by controlling cell death. The mechanism used by Hha is the control of translation via the availability of rare codon tRNAs which reduces fimbriae production and activates prophage lytic genes. Therefore, Hha acts as a toxin in conjunction with co-transcribed YbaJ (TomB) that attenuates Hha toxicity

The sphingosine-1-phosphate receptor-1 antagonist, W146, causes early and short-lasting peripheral blood lymphopenia in mice

Agonists of the sphingosine-1-phosphate (S1P) receptors, like fingolimod (FTY720), are a novel class of immunomodulators. Administration of these compounds prevents the egress of lymphocytes from primary and secondary lymphoid organs causing peripheral blood lymphopenia. Although it is well established that lymphopenia is mediated by S1P receptor type 1 (S1P1), the exact mechanism is still controversial. The most favored hypothesis states that S1P1 agonists cause internalization and loss of the cell surface receptor on lymphocytes, preventing them to respond to S1P. Hence, S1P1 agonists would behave in vivo as functional antagonists of the receptor. For this hypothesis to be valid, a true S1P1 antagonist should also induce lymphopenia. However, it has been reported that S1P1 antagonists fail to show this effect, arguing against the concept. Our study demonstrates that a S1P1 antagonist, W146, induces a significant but transient blood lymphopenia in mice and a parallel increase in CD4+ and CD8+ lymphocytes in lymph nodes. Treatment with W146 also causes the accumulation of mature T cells in the medulla of the thymus and moreover, it induces lung edema. We show that both the S1P1 antagonist and a S1P1 agonist cause lymphopenia in vivo in spite of their different effects on receptor expression in vitro. Although the antagonist purely blocks the receptor and the agonist causes its disappearance from the cell surface, the response to the endogenous ligand is prevented in both cases. Our results support the hypothesis that lymphopenia evoked by S1P1 agonists is due to functional antagonism of S1P1 in lymphocytes.Gema Tarrasón, Mariona Aulí, Sanam Mustafa, Vladislav Dolgachev, Maria Teresa Domènech, Neus Prats, María Domínguez, Rosa López, Nuria Aguilar, Marta Calbet, Mercè Pont, Graeme Milligan, Steven L. Kunkel, Nuria Godessar

Pharmacological blockade of CCR1 ameliorates murine arthritis and alters cytokine networks in vivo

The chemokine receptor CCR1 is a potential target for the treatment of rheumatoid arthritis. To explore the impact of CCR1 blockade in experimental arthritis and the underlying mechanisms, we used J-113863, a non-peptide antagonist of the mouse receptor. Experimental approach: Compound J-113863 was tested in collagen-induced arthritis (CIA) and three models of acute inflammation; Staphylococcus enterotoxin B (SEB)-induced interleukin-2 (IL-2), delayed-type hypersensitivity (DTH) response, and lipopolysaccharide (LPS)-induced tumour necrosis factorΑ (TNFΑ) production. In the LPS model, CCR1 knockout, adrenalectomised, or IL-10-depleted mice were also used. Production of TNFΑ by mouse macrophages and human synovial membrane samples in vitro were also studied. Key results: Treatment of arthritic mice with J-113863 improved paw inflammation and joint damage, and dramatically decreased cell infiltration into joints. The compound did not inhibit IL-2 or DTH, but reduced plasma TNFΑ levels in LPS-treated mice. Surprisingly, CCR1 knockout mice produced more TNFΑ than controls in response to LPS, and J-113863 decreased TNFΑ also in CCR1 null mice, indicating that its effect was unrelated to CCR1. Adrenalectomy or neutralisation of IL-10 did not prevent inhibition of TNFΑ production by J-113863. The compound did not inhibit mouse TNFΑ in vitro, but did induce a trend towards increased TNFΑ release in cells from synovial membranes of rheumatoid arthritis patients. Conclusions and implications: CCR1 blockade improves the development of CIA, probably via inhibition of inflammatory cell recruitment. However, results from both CCR1-deficient mice and human synovial membranes suggest that, in some experimental settings, blocking CCR1 could enhance TNF production. British Journal of Pharmacology (2006) 149 , 666–675. doi:Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75213/1/sj.bjp.0706912.pd