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  • br Results and Discussion br Conclusion What we


    Results and Discussion
    Conclusion What we have shown here is that highly specific antagonist can be a very useful tool, can mimic gene KO experiments or even further dissect receptor function. We just took ETA antagonist as an example and found that ETA mediates physiological closure of DA which would not be observed because of the immediate lethality in the gene KO experiments. And finally, we showed potential utilities of the ETA specific antagonists in tocolysis with NSAIDs.
    Background: The endothelin A receptor (ETA) mediates tumourigenic signals in colorectal cancer (CRC). The ETA ligand, endothelin-1 (ET-1), stimulates not only cancer AG014699 but also surrounding fibroblasts and may promote the creation of a supporting tumour stroma. Aim: To identify ET-1 regulated genes associated with oncogenic pathways in colonic fibroblasts. Methods: Micro-array analysis following 4h /INS;ET-1 stimulation of colonic fibroblast strains (isolated from patients undergoing resection for CRC, n=4) identified differentially expressed genes (n=19) at significant levels. Three were investigated further: COLXI, AML-1, and /INS;EGFR (collagen type-XI; acute myeloid leukemia-1; epidermal growth-factor receptor). Quantitative RT-PCR (qRT-PCR) and immunoblotting evaluated AML-1 and COLX expression levels, following treatment with ET-1 and/or receptor antagonists (ETA: BQ123, ZD4054; ETB: BQ788). ETA and ETB regulation of EGFR was investigated by gene silencing (siRNA); these assays and ET-1 regulation of EGFR over 24h /INS;were evaluated by qRT-PCR. Results: ET-1 stimulated expression of AML-1 and COLXI at both gene (>1.5-fold; p<0.01) and protein (p<0.05) levels; stimulation was inhibited by ETA, but not by /INS;ETB, antagonism (AML-1: /INS;75.1–/INS;77.1% by BQ123, ZD4054; COLXI: /INS;65.1% by ZD4054; p<0.05). EGFR expression demonstrated a biphasic increase at 4h /INS;and 24h /INS;(3.8-fold; 4.5-fold). Silencing ETA, but not ETB, returned EGFR levels to control. Conclusions: ETA antagonism has potential for targeting oncogenic pathways: AML-1 is linked to c-Jun N-terminal kinase which inhibits apoptosis/promotes proliferation; and abnormal TGF-β (transforming growth-factor-beta) signalling. COLXI is linked to CRC tumourogenesis. The ET-1-stimulated biphasic EGFR response and ETA antagonism have not been reported before in CRC. These findings identify mechanisms by which ETA promotes tumourigenesis and support addition of ZD4054 to existing EGFR antagonism therapy.
    Introduction Diabetes mellitus is associated with a considerable risk for cardiovascular complications such as peripheral arterial disease, coronary artery disease and myocardial infarction (Moreno and Fuster, 2004). There are observations suggesting that endothelial dysfunction plays an important role in the early development of vascular complications in diabetes (Creager et al., 2003). Presence of endothelial dysfunction is also an independent risk factor for future cardiovascular events in patients with coronary artery disease (Lerman and Zeiher, 2005). Endothelial dysfunction is characterized by reduced bioavailability of the vasodilator and anti-inflammatory molecule nitric oxide. Another factor of potential importance for endothelial dysfunction in type 2 diabetes is endothelin-1 (ET-1) which exerts potent vasoconstrictor and pro-inflammatory actions via activation of the two receptors, ETA and ETB. Both receptors are present on vascular smooth muscle cells mediating vasoconstriction, whilst the ETB receptor is also present on endothelial cells mediating vasodilatation. Elevated levels of ET-1 are found in patients with type 2 diabetes (Takahashi et al., 1990). Due to the dynamics of the expression and function of the two ET receptors described above, the effect of ET receptor blockade may vary depending on the type and stage of pathological condition. Whilst the ETA receptor inevitably causes vasoconstriction and exerts pro-oxidative actions, ETB receptor stimulation may cause vasodilatation in healthy subjects due to the stimulation of NO production or it may cause vasoconstriction in patients with atherosclerosis due to upregulation of the ETB receptor on vascular smooth muscle cells (Pernow et al., 2012). The potential beneficial or detrimental effect of dual ETA/ETB receptor blockade in comparison with selective ETA receptor blockade has therefore been a matter of much discussion. A recent clinical study demonstrated that the dual ET receptor antagonist bosentan improves microvascular endothelial function in patients with type 2 diabetes which suggests beneficial effect of dual receptor blockade (Rafnsson et al., 2012). However, a head to head comparison between selective and dual receptor blockade in patients with coronary artery disease and type 2 diabetes has to the best of our knowledge not been performed. It therefore is of importance in detail to clarify the effect of selective ETA vs. dual ETA/ETB receptor blockade on endothelial function in patients with type 2 diabetes and vascular complications. The hypothesis was that dual ETA/ETB results in greater vasodilator effect and improvement in endothelium-dependent vasodilatation than selective ETA receptor blockade.