P479Extracellular S100A4 induces arterial smooth muscle cell activation in a RAGE-dependent manner

Background: It has been proposed that smooth muscle cells (SMCs) from the arterial wall are heterogeneous and that only a subset of medial SMCs are prone to accumulate into the intima leading to atheromatous plaque formation. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as being a marker of the R-SMCs in vitro and of intimal SMCs, both in pig and human. S100A4 is a Ca2+-binding protein that can also be secreted; it has extracellular functions probably via the receptor for advanced glycation end products (RAGE). Purpose: Explore the role of S100A4 in SMC phenotypic change, a phenomenon characteristic of atherosclerotic plaque formation. Methods and Results: Transfection of a human S100A4-containing plasmid in spindle-shaped (S) SMCs (devoid of S100A4) led to approximately 10% of S100A4-overexpressing SMCs, S100A4 release, and a transition towards a R-phenotype of the whole SMC population. Furthermore treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a phenotype typical of the R-SMCs, which was associated with decreased SMC differentiation markers, increased proliferation and migration, as well as induced proteolytic activity through activation of urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMP-1,-2, -3, and -9) and their inhibitors (TIMP-1). Furthermore, extracellular S100A4 yielded activation of NF-kB in a RAGE-dependent manner. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulation of SMC differentiation markers. In contrast, silencing of S100A4 mRNA in R-SMCs did not change the level of extracellular S100A4 nor SMC morphology in spite of decreased proliferative activity. Conclusions: Our results indicate that SMC phenotypic changes are essentially dependent on extracellular S100A4 activity. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosi

304Nuclear targeting apelin induces phenotypic transition of vascular smooth muscle cells

Background: Apelin, and its receptor APJ, are a peptidic system playing a crucial role in vascular diseases. However, the role of apelin in atherogenesis and smooth muscle cell (SMC) proliferation remains unclear. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. S100A4 is a marker of R-SMCs in vitro and of intimal SMCs, both in pig and human. S100A4 is a Ca2+-binding protein that can also be secreted; it has extracellular functions probably via the receptor for advanced glycation end products (RAGE). Purpose: Investigate the effects of apelin on SMC phenotypic transition and S100A4 expression and release. Methods and Results: We observed that apelin was highly expressed in R-SMCs particularly in their nucleus. P-SORT software analysis of preproapelin sequence suggested that N-terminal truncated apelin may target the nucleus, and we confirmed this in SMCs by overexpression of mutated preproapelin-His-tag. Transfection of mutated preproapelin-His-tag encoding plasmid in differentiated S-SMCs induced a transition towards a R-phenotype associated with increased proliferative activity, downregulation of SMC differentiation markers (i.e. alpha-smooth muscle actin), and increased nuclear expression and release of S100A4. In contrast, transfection of S-SMCs with wild type preproapelin-His-tag encoding plasmid did not induce nuclear targeting of Apelin or S100A4, and did not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB, known to induce a transition to the R-phenotype, yielded nuclear targeting of both apelin and S100A4. In vivo, Apelin was expressed in SMC nuclei of stent-induced intimal thickening while its expression in the media was mainly cytoplasmic. Conclusions: Our results suggest that nuclear targeting of apelin in SMCs acts on S100A4 expression and release, cell proliferation and differentiation. The pathophysiological consequences of this retargeting could be instrumental in the understanding of artherosclerosi

Evidence for modulation of pericryptal sheath myofibroblasts in rat descending colon by Transforming Growth Factor β and Angiotensin II.

BACKGROUND: Absorption of water and Na(+) in descending colonic crypts is dependent on the barrier function of the surrounding myofibroblastic pericryptal sheath. Here the effects of high and low Na(+) diets and exposure to whole body ionising radiation on the growth and activation of the descending colonic pericryptal myofibroblasts are evaluated. In addition the effect of a post-irradiation treatment with the angiotensin converting enzyme inhibitor Captopril was investigated. METHODS: The levels of Angiotensin II type 1 receptor (AT1), ACE, collagen type IV, transforming growth factor-β type 1 receptor (TGF-βR1), OB cadherin and α-smooth muscle actin in both descending colon and caecum were evaluated, using immunocytochemistry and confocal microscopy, in rats fed on high and low Na(+) diets (LS). These parameters were also determined during 3 months post-irradiation with 8Gy from a (60)Co source in the presence and absence of the angiotensin converting enzyme inhibitor, Captopril. RESULTS: Increases in AT1 receptor (135.6% ± 18.3, P < 0.001); ACE (70.1% ± 13.1, P < 0.001); collagen type IV (49.6% ± 15.3, P < 0.001); TGF-β1 receptors (291.0% ± 26.5, P < 0.001); OB-cadherin (26.3% ± 13.8, P < 0.05) and α-smooth muscle actin (82.5% ± 12.4, P < 0.001) were observed in the pericryptal myofibroblasts of the descending colon after LS diet. There are also increases in AT1 receptor and TGF-β1 receptor, smooth muscle actin and collagen type IV after irradiation. Captopril reduced all these effects of irradiation on the pericryptal sheath and also decreased the amount of collagen and smooth muscle actin in control rats (P < 0.001). CONCLUSIONS: These results demonstrate an activation of descending colonic myofibroblasts to trophic stimuli, or irradiation, which can be attenuated by Captopril, indicative of local trophic control by angiotensin II and TGF-β release

P2 receptors in atherosclerosis and postangioplasty restenosis

Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction [Ross (Nature 362:801–09, 1993); Fuster et al. (N Engl J Med 326:242–50, 1992); Davies and Woolf (Br Heart J 69:S3–S11, 1993)]. Extracellular nucleotides that are released from a variety of arterial and blood cells [Di Virgilio and Solini (Br J Pharmacol 135:831–42, 2002)] can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells (SMC), which are known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis [Lafont et al. (Circ Res 76:996–002, 1995)]. In addition, P2 receptors mediate many other functions including platelet aggregation, leukocyte adherence, and arterial vasomotricity. A direct pathological role of P2 receptors is reinforced by recent evidence showing that upregulation and activation of P2Y2 receptors in rabbit arteries mediates intimal hyperplasia [Seye et al. (Circulation 106:2720–726, 2002)]. In addition, upregulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model [Carpenter et al. (Stroke 32:516–22, 2001)] and in coronary artery of diabetic dyslipidemic pigs [Hill et al. (J Vasc Res 38:432–43, 2001)]. It has been proposed that upregulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis [Elmaleh et al. (Proc Natl Acad Sci U S A 95:691–95, 1998)]. Therefore, particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels, which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty

Mitochondrial localization of glucocortocoid receptor in glial (Muller) cells in the salamander retina

Glucocorticoid hormones regulate the transcription of nuclear genes by way of their receptors. In addition, these hormones modulate mitochondrial gene transcription by mechanisms that remain poorly understood. Using immunofluorescence labeling in isolated Muller and photoreceptor cells and in intact salamander retina, we found that the glucocorticoid receptor (GR) is localized in both cell types. Confocal laser scanning microscopy and double staining with cytochrome oxidase (COX) showed that GR is localized in the mitochondria of Muller cells, but not in the mitochondria of photoreceptors. GR also colocalizes with glutamine synthetase (GS) in the cytoplasm of Muller cells. GR is also localized in the microvilli of the distal process of Muller cells and in the synaptic terminal of photoreceptors. Pre-incubation of Muller cells with 1 muM dexamethasone (DEX) for 7 h led to greater than 50% inhibition of the glutamate-induced increase in mitochondrial NADH. This late effect of glucocorticoids on glutamate metabolism could be ascribed, in part, to a direct action of steroid hormones on mitochondrial. metabolism. (C) 2003 Wiley-Liss, Inc