Locally applied cilostazol suppresses neointimal hyperplasia by inhibiting tenascin-C synthesis and smooth muscle cell proliferation in free artery grafts

AbstractObjectiveAccumulation of smooth muscle cells and extracellular matrix in the intima of artery bypass grafts induces neointimal hyperplasia, resulting in graft failure. We investigated the inhibitory effect of locally applied cilostazol, an inhibitor of cyclic adenosine monophosphate phosphodiesterase III, on neointimal hyperplasia and the role of tenascin-C synthesis and smooth muscle cell proliferation in free artery grafts.Methods and resultsWe established a distal anastomotic stricture model of free artery graft stenosis using rat abdominal aorta. In this model, neointimal hyperplasia was observed not only in the distal anastomotic site but also in the graft body at postoperative day 14 and was markedly progressed at day 28. Strong expression of tenascin-C was found in the media and neointima of the graft body. When cilostazol was locally administered around the graft using Pluronic gel, neointimal hyperplasia of the graft was significantly suppressed in comparison with gel-treated control graft. The mean neointima/media area ratio was reduced by 86.6% for the graft body and by 75.8% for the distal anastomotic site versus the control. Cilostazol treatment decreased cell proliferation and tenascin-C expression in the neointima. In an in vitro experiment using cultured smooth muscle cells isolated from rat aorta, cilostazol completely suppressed the tenascin-C mRNA expression induced by platelet-derived growth factor-BB.ConclusionA single topical administration of cilostazol may suppress neointimal hyperplasia by inhibiting cell proliferation and tenascin-C synthesis in free artery grafts, presenting the potential for clinical use in vascular surgery

Cardiac tamponade due to rupture of a giant coronary artery aneurysm with a coronary arteriovenous fistula: a case report

Abstract Background Coronary artery aneurysm (CAA) is defined as dilatation exceeding 1.5 times the width of the normal adjacent coronary artery segments. CAA usually causes few symptoms, and rupture is rare, but can be lethal due to cardiac tamponade when it does occur. Case presentation A 79-year-old woman presented with presyncope and back pain. Emergency surgery was performed based on a diagnosis of cardiac tamponade due to either rupture of coronary arteriovenous fistula or CAA. At surgery, a rupture site was located on the wall of the giant CAA, with a diameter of 55 mm, originating from the ostium of the right coronary artery. Suture closure of the inflow and outflow of the aneurysm was performed, and the aneurysmal cavity was obliterated by multiple sutures. The patient made an uneventful recovery and was discharged from hospital on postoperative day 13. Conclusion On the basis of this case, we propose considering rupture of a CAA as one of the causes of cardiac tamponade

Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver.

The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS) in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2'-5' oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity

Heterozygous knockout of Bile salt export pump ameliorates liver steatosis in mice fed a high-fat diet.

The incidence of nonalcoholic steatohepatitis (NASH) is increasing worldwide, including in Asian countries. We reported that the hepatic expression of bile salt export pump (BSEP) was downregulated in patients with NASH, suggesting that BSEP is involved in the pathogenesis of NASH. To identify the underlying mechanism, we analyzed Bsep heterozygous knock-out (Bsep+/- mice) and wild-type (WT) C57BL/6J mice fed a high-fat diet (HFD) (32.0% animal fat) or normal diet. We examined histological changes, levels of hepatic lipids and hepatic bile acids, and expression of genes related to bile acid and cholesterol metabolism. HFD-fed Bsep+/- mice exhibited milder hepatic steatosis and less weight gain, compared to HFD-fed WT mice. The concentrations of total bile acid, triglycerides, and cholesterols were reduced in the liver of HFD-fed Bsep+/- mice. Regarding hepatic bile acid metabolism, the expression levels of Farnesoid X receptor (Fxr) and Multidrug resistance-associated protein 2 were significantly upregulated in HFD-fed Bsep+/- mice, compared to HFD-fed WT mice. Furthermore, several alterations were observed in upstream cholesterol metabolism in the liver. The expression levels of bile acid metabolism-related genes were also altered in the intestine of HFD-fed Bsep+/- mice. In conclusion, HFD-fed Bsep+/- mice exhibited significant alterations of the expression levels of genes related to bile acid and lipid metabolism in both the liver and ileum, resulting in alleviated steatosis and less weight gain. These results suggest the importance of BSEP for maintenance of bile acid and cholesterol metabolism. Further investigations of the involvement of BSEP in the pathogenesis of NASH will provide greater insight and facilitate the development of novel therapeutic modalities

Suppressed activation of STAT3 in NS5A-transgenic mice.

<p>(A) Phosphorylation levels of STAT3 were determined by immunoblotting with phospho-specific antibodies in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice 6 h after injection with LPS or normal saline. (B) Liver tissue sections derived from LPS-injected transgenic (Tg) and nontransgenic (NT) mice were immunohistologically stained with an anti-phospho-STAT3 antibody. (C) DNA-binding activity of STAT3 was determined by EMSA. Shifted bands corresponding to DNA-bound STAT3 were shown (left panel). The specificity of the band was examined by adding 100-fold amount of non-labeled, identical (I) or mutated (M) oligonucleotides before the addition of labeled oligonucleotides (right panel).</p

Expression of NS5A in transgenic mice.

<p>(A) Expression levels of NS5A protein in liver tissues of NS5A-transgenic (Tg) and non-transgenic (NT) mice were determined by immunoblotting with mouse anti-NS5A antibody. As a positive control (PC), lysates of the HCV subgenomic replicon cells were used. The arrow represents the NS5A protein. The arrowhead indicates a nonspecific band observed only in mouse tissues. (B) mRNA expression levels of NS5A in the liver of transgenic mice and HCV-infected patients were determined using quantitative real-time PCR. The levels were standardized to the β-actin gene. The median value of human samples was defined as 1 in Y-axis. (C) Liver tissue sections of the 6-month-old NS5A-transgenic (Tg) and non-transgenic (NT) mice were histologically examined with H&E staining.</p

Suppressed TLR signaling in the livers of LPS-injected NS5A-transgenic mice.

<p>(A) Phosphorylation levels of ERK, IκB-α, and IRF7 were determined by immunoblotting with phospho-specific antibodies in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice injected with LPS. (B) Interaction between IRAK1 and TRAF6 was examined using an immunoprecipitation experiment. Liver tissue lysates of NS5A-transgenic (Tg) and non-transgenic (NT) mice were immunoprecipitated with anti-TRAF6 antibody and the immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with anti-IRAK1 and anti-TRAF6 antibodies. Whole cell lysates (WCL) were similarly subjected to immunoblotting with anti-IRAK1 and anti-vinculin antibodies.</p