An inhibitor of thrombin activated fibrinolysis inhibitor (TAFI) can reduce extracellular matrix accumulation in an in vitro model of glucose induced ECM expansion.

Chronic kidney disease (CKD) is characterised by the pathological accumulation of extracellular matrix (ECM) proteins leading to progressive kidney scarring via glomerular and tubular basement membrane expansion. Increased ECM synthesis and deposition, coupled with reduced ECM breakdown contribute to the elevated ECM level in CKD. Previous pre-clinical studies have demonstrated that increased plasmin activity has a beneficial effect in the protein overload model of CKD. As plasmin activation is downregulated by the action of the thrombin activated fibrinolytic inhibitor (TAFI), we tested the hypothesis that inhibition of TAFI might increase plasmin activity and reduce ECM accumulation in an in vitro model of glucose induced ECM expansion. Treatment of NRK52E tubular epithelial cells with increasing concentrations of glucose resulted in a 40% increase in TAFI activity, a 38% reduction in plasmin activity and a subsequent increase in ECM accumulation. In this model system, application of the previously reported TAFI inhibitor UK-396082 [(2S)-5-amino-2-[(1-n-propyl-1H-imidazol-4-yl)methyl]pentanoic acid] caused a reduction in TAFI activity, increased plasmin activity and induced a parallel decrease in ECM levels. In contrast, RNAi knockdown of plasmin resulted in an increase in ECM levels. The data presented here indicate that high glucose induces TAFI activity, inhibiting plasmin activation which results in elevated ECM levels in tubular epithelial cells. The results support the hypothesis that UK-396082 is able to reduce TAFI activity, normalising plasmin activity and preventing excess ECM accumulation suggesting that TAFI inhibition may have potential as an anti-scarring strategy in CKD.

Cellular exchange in an endometriosis-adhesion model using GFP transgenic mice.

BACKGROUND: Endometriosis is a debilitating disease that affects women of reproductive age and may lead to impaired fertility. Cell attachment, invasion of the underlying tissue, and vascular ingrowth are important processes in endometrial lesion development. However, the degree of cellular exchange between host peritoneum and endometrial tissue is unclear. METHODS: An experimental endometriosis model was employed whereby uterine horn fragments from wild-type mice were implanted into genetically identical eGFP (enhanced green fluorescent protein) host mice and vice versa. Hormone sensitivity of the ectopic lesions was assessed and cellular exchange determined histologically. RESULTS: White cyst-like lesions developed from implanted fibrin-rich fragments by day 7. Lesions consisted of a well-developed stroma with glandular and luminal epithelium. Both ovariectomy and treatment with a GnRH agonist, leuprorelin, resulted in the suppression of ectopic lesion growth, whereas estradiol treatment increased the size of the ectopic lesion (4 mice per group on day 14). Ingrowth and outgrowth of blood vessels was apparent as well as the exchange of cells between host peritoneum and lesion. CONCLUSION: These findings support the proposal that there is a close cellular interplay between host peritoneum and ectopic tissue and the suitability of this mouse model to study these interactions.

In vitro and in vivo characterization of PF-04418948, a novel, potent and selective prostaglandin EP2 receptor antagonist.

BACKGROUND AND PURPOSE: Studies of the role of the prostaglandin EP(2) receptor) have been limited by the availability of potent and selective antagonist tools. Here we describe the in vitro/in vivo pharmacological characterization of a novel EP(2) receptor antagonist, PF-04418948 (1-(4-fluorobenzoyl)-3-{[(6-methoxy-2-naphthyl)oxy]methyl} azetidine-3-carboxylic acid). EXPERIMENTAL APPROACH: Functional antagonist potency was assessed in cell-based systems expressing human EP(2) receptors and native tissue preparations from human, dog and mouse. The selectivity of PF-04418948 was assessed against related receptors and a panel of GPCRs, ion channels and enzymes. The ability of PF-04418948 to pharmacologically block EP(2) receptor function in vivo was tested in rats. KEY RESULTS: PF-04418948 inhibited prostaglandin E(2)(PGE(2))-induced increase in cAMP in cells expressing EP(2) receptors with a functional K(B) value of 1.8 nM. In human myometrium, PF-04418948 produced a parallel, rightward shift of the butaprost-induced inhibition of the contractions induced by electrical field stimulation with an apparent K(B) of 5.4 nM. In dog bronchiole and mouse trachea, PF-04418948 produced parallel rightward shifts of the PGE(2)-induced relaxation curve with a K(B) of 2.5 nM and an apparent K(B) of 1.3 nM respectively. Reversal of the PGE(2)-induced relaxation in the mouse trachea by PF-04418948 produced an IC(50) value of 2.7 nM. Given orally, PF-04418948 attenuated the butaprost-induced cutaneous blood flow response in rats. PF-04418948 was selective for EP(2) receptors over homologous and unrelated receptors, enzymes and channels. CONCLUSIONS AND IMPLICATIONS: PF-04418948 is an orally active, potent and selective surmountable EP(2) receptor antagonist that should aid further elaboration of EP(2) receptor function.

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells.

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27KiP1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.

Phosphorylation and activation of p70s6k by PDK1.

Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.