The organ culture method has previously shown to

The organ culture method has previously shown to be a suitable model for investigations of receptor upregulation on vascular smooth muscle cells (Adner et al., 1996). In our study, the organ culture method was applied in order to examine whether LPS from P.g. was capable of altering the gene expression of the contractile ETB receptors on vascular smooth muscle cells. Previous studies have demonstrated that endothelial function is slightly reduced during organ culture, and the dilatory function of ETB receptors on the endothelium has been suggested to be down-regulated (Nilsson et al., 2008c) as observed in cardiovascular diseases. In agreement with previous studies on ETB receptor upregulation using the organ culture procedure (Adner et al., 1996, Adner et al., 1998, Adner et al., 2001, Eskesen and Edvinsson, 2006, Nilsson et al., 2008b, Nilsson et al., 2008c, Wackenfors et al., 2004), the present study demonstrated a strong vasoconstriction induced by ETB receptors in coronary LY 2389575 hydrochloride australia after 24h organ culture as compared to freshly isolated coronary arteries. This was associated with enhanced expression of ETB receptor protein on smooth muscle cells of cultured coronary arteries as confirmed by immunohistochemistry. Furthermore, our study revealed an ability of LPS to increase the sensitivity of the contractile response to ETB receptor stimulation. Strikingly, our results revealed an important role for endothelium in LPS-treated vessels since LPS induced a significant increase in the sensitivity of contractile responses towards S6c only in coronary arteries with an intact endothelium. This may indicate an interaction between the endothelium and LPS. Despite functional studies demonstrating a significant LPS-mediated increase in the contractile sensitivity of endothelium-intact cultured coronary arteries to the selective ETB receptor agonist S6c, immunohistochemistry could not confirm further enhancement of the ETB receptor protein level by LPS after 24h organ culture. To further verify the presence of ETB receptors on the smooth muscle cells of cultured coronary arteries with or without presence of LPS in the culturing medium, the selective competitive ETB receptor antagonist (BQ788) was applied in these experiments. In both vessel groups, BQ788 induced a significant concentration-dependent rightward shift in the log S6c – response curve, which is indicative of the presence of contractile ETB receptors in both groups of cultured vessels. As far as the contractile response to ETA receptors is concerned, no significant difference in ETA-mediated contraction was observed after desensitization of the ETB receptors with S6c, comparing fresh and cultured arteries. The presence of ETA receptors on smooth muscle cells was also verified by immunohistochemistry. However, there was no significant difference between the three groups of coronary arteries (i.e. freshly isolated, 24h organ culture with LPS, and 24h organ culture without LPS) regarding ETA receptor expression, correlating well with the functional studies of ETA receptor. In general, the results from our study are in concert with a previous study showing a clear association between periodontitis and endothelial dysfunction (Tonetti et al., 2007). Thus, periodontitis patients showed a reduced vasodilatory ability due to endothelial dysfunction, the reaction being normalized after periodontal treatment. Previously published studies have shown ETB receptor upregulation in severe pathophysiological conditions such as ischemic stroke (Stenman et al., 2002), ischemic heart disease (Wackenfors et al., 2004, Wendel-Wellner et al., 2002), atherosclerosis (Dagassan et al., 1996), congestive heart failure (Cannan et al., 1996), subarachnoid haemorrhage (Hansen-Schwartz, 2004, Roux et al., 1995) and this phenomenon may partly be explained by a down-regulation of endothelial ETB receptors that normally induce vasodilatation through formation and release of nitric oxide, or an upregulation of smooth muscle ETB receptors that mediate vasoconstriction. Recently, two major signalling pathways such as protein kinase C (PKC) and MAPK have been suggested to be involved in the up-regulation of ETB receptors in human internal mammary arteries after the organ culture procedure (Nilsson et al., 2008a). Recent studies have revealed that mammalian Toll-like receptors (TLRs) are key proteins for recognizing pathogen-associated molecules such as LPS from gram-negative bacteria to induce an inflammatory response (Kopp and Medzhitov, 1999). TLRs initiate and maintain host defenses and inflammation, and directly contribute to diseases such as atherosclerosis. The interaction of LPS with TLRs can activate the mitogen activating protein kinase (MAPK) signalling cascade and thereby transduce important intracellular signaling mechanisms such as gene expression (Pan, 2004). These findings may be of relevance to coronary artery disease, as it has been shown that TLR2 deficiency reduces atherosclerosis in ApoE−/−mice (Mullick et al., 2005), while TLR2 stimulation accelerates atherosclerosis (Schoneveld et al., 2005).