In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

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The supraoptic nucleus (SON) of the hypothalamus contains cell bodies of two different populations of neurosecretory cells, vasopressin and oxytocin neurones. Release of vasopressin and oxytocin in the neurohypophysis is controlled by the specific electrical activity of these neurones, which is regulated by synaptic inputs into the SON mediated by various neurotransmittersÏneuromodulators, such as GABA and glutamate (Decavel & Van den Pol, 1990;Van den Pol et al. 1990;Wuarin & Dudek, 1993). A1 noradrenergic neurones originating from the ventrolateral

Background: A host receptor has not yet been identified for glycerol monomycolate (GroMM), an immunostimulatory lipid of mycobacteria. Results: GroMM recognition occurred in cell transfectants expressing human, but not mouse Mincle. Human Mincle transgenic mice acquired the ability to respond to GroMM. Conclusion: GroMM is a ligand for human Mincle. Significance: The molecular basis underlying the innate immune recognition of GroMM has been elucidated.

Background and purpose: Proteinase-activated receptor 2 (PAR2) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR2 antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR2 antagonists and demonstrate inhibitory effects on PAR2-mediated intracellular signalling pathways and vascular responses. Experimental approach: The peptide-mimetic compound library based on the structures of PAR2 agonist peptides were screened for inhibition of PAR2-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models.

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To determine the binding characteristics of a highly potent agonist for protease‐activated receptor‐2 (PAR2), 2‐furoyl‐Leu‐Ile‐Gly‐Arg‐Leu‐amide (2‐furoyl‐LIGRL‐NH2), whole‐cell binding assays were performed utilising a radioactive ligand, [3H]2‐furoyl‐LIGRL‐NH2. Specific binding of [3H]2‐furoyl‐LIGRL‐NH2 was observed in NCTC2544 cells, dependent upon PAR2 expression, and competitively displaced by the addition of unlabeled PAR2 agonists. Scatchard analysis of specific saturation binding suggested a single binding site, with Kd of 122±26.1 nM and a corresponding Bmax of 180±6 f mol in 3.0 × 105 cells. The relative binding affinities of a series of modified PAR2 agonist peptides obtained from competition studies paralleled their relative EC50 values for Ca2+ mobilisation assays, indicating improved binding affinities by substitution with 2‐furoyl at the N‐terminus serine. Pretreatment of cells with trypsin reduced specific binding of [3H]2‐furoyl‐LIGRL‐NH2, demonstrating direct competition between the synthetic agonist peptide and the proteolytically revealed tethered ligand for the binding site of the receptor. In HCT‐15 cells endogenously expressing PAR2, the binding of [3H]2‐furoyl‐LIGRL‐NH2 was displaced by addition of unlabeled ligands, Ser‐Leu‐Ile‐Gly‐Lys‐Val (SLIGKV‐OH) or 2‐furoyl‐LIGRL‐NH2. The relative binding affinity of 2‐furoyl‐LIGRL‐NH2 to SLIGKV‐OH was comparable to its relative EC50 value for Ca2+ mobilisation assays. The binding assay was successfully performed in monolayers of PAR2 expressing NCTC2544 and human umbilical vein endothelial cells (HUVEC), in 96‐ and 24‐well plate formats, respectively. These studies indicate that [3H]2‐furoyl‐LIGRL‐NH2 binds to human PAR2 at its ligand‐binding site. The use of this radioligand will be valuable for characterising chemicals that interact to PAR2. British Journal of Pharmacology (2005) 145, 255–263. doi: