A novel tissue treatment to reduce mineralization of bovine pericardial heart valves.

OBJECTIVE: With the increasing use of bioprostheses worldwide, continuous efforts have been made to improve tissue durability. We introduce a new treatment for bovine pericardium combining octanediol-ethanol based phospholipid removal with taurine-based glutaraldehyde neutralization and storage in an aldehyde-free solution (FREE). METHODS: Treated tissues were evaluated by mechanical and biochemical characterization, phospholipid content, aldehyde levels, cell cultures on pericardial samples (L929 fibroblasts and human umbilical vein endothelial cells), rat subcutaneous implantations, and long-term juvenile sheep mitral valve implantations (n = 3). Comparisons were made to glutaraldehyde-fixed bovine pericardium or to samples from commercially available biological valves (ie, Trifecta [St Jude Medical, Saint Paul, Minn] and Perimount Magna Ease [Edwards Lifesciences, Irvine, Calif]). RESULTS: FREE-treated pericardium had similar mechanical strength and biochemical properties as commercially available valves. Compared with glutaraldehyde-only samples, FREE-treated samples showed lower phospholipid levels (P < .01), significantly better growth of L929 fibroblasts, and lower calcification levels in rat subcutaneous implants (P < .01). Compared with samples from Linx- (Trifecta) and ThermaFix-treated (Perimount Magna Ease) valves, similar low levels of phospholipids were observed as were similar low calcification levels in subcutaneous implants, but tissue extractions from FREE-treated samples showed the lowest levels of extracted aldehydes (P < .01). Mitral implants of FREE-treated valves in juvenile sheep had excellent hemodynamic behavior without any sign of degeneration or calcification at 5 months. CONCLUSIONS: The new FREE treatment combines an adequate phospholipid reduction and aldehyde neutralization with storage in an aldehyde-free solution. This combination enhances the anticalcification properties and may thereby improve long-term durability of the tissue.

IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism.

Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.

CCL16 activates an angiogenic program in vascular endothelial cells.

Besides regulating leukocyte trafficking in normal and injured tissues, several chemokines may positively or negatively regulate angiogenesis. Here we report that CCL16 activates an angiogenic program in vascular endothelial cells by activating CCR1. CCL16 induces dose-dependent random and directional migration of endothelial cells isolated from large vessels and liver capillaries without inducing their proliferation. It also promotes endothelial differentiation into capillary-like structures in an in vitro assay and is angiogenic in the chick chorionallantoic membrane. These angiogenic activities are neutralized by a specific antibody against CCL16. The direct angiogenic activity of CCL16 is further amplified by its ability to prime endothelium to a mitogen signal induced by vascular endothelial growth factor A and to raise their basal production of CXCL8 and CCL2, 2 other angiogenic chemokines. BX471 (R-N-[5-chloro-2-[2-[4(4-fluorophenyl) methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl] urea hydrochloric acid salt), a CCR1 antagonist, inhibits angiogenic properties of CCL16, whereas blocking of CCR8 or desensitizing CCR2, which are both well known receptors for CCL16, did not abolish endothelial activation. CCL16 may be specifically cross-linked to CCR1 expressed on endothelial cells. The largely restricted CCL16 expression in the liver suggests that this chemokine may play a role in hepatic vascular formation during development and in angiogenesis associated to hepatic diseases.

IL-12 regulates an endothelial cell-lymphocyte network: effect on metalloproteinase-9 production.

IL-12 is key cytokine in innate immunity and participates in tumor rejection by stimulating an IFN-gamma-mediated response characterized by CD8(+) mediated-cytotoxicity, inhibition of angiogenesis, and vascular injury. We previously demonstrated that activated lymphocytes stimulated with IL-12 induced an angiostatic program in cocultured vascular endothelial cells. In this study, we have extended this observation showing that a reciprocal modulation of cellular responses occurs. Actually, the presence of endothelial cells enhanced the inhibitory effect of IL-12 on metalloproteinase-9 expression in activated PBMC as well as their ability to transmigrate across an extracellular matrix. IL-12 triggered intracellular signaling, as indicated by STAT-1 activation, appeared to mainly operative in activated CD4 (+) cells challenged with IL-12, but it was also initiated in CD8(+) lymphocytes in the presence of endothelial cells. On the other hand, stimulated PBMC reduced the expression and the activity of metalloproteinase-9, up-regulated that of tissue inhibitor metalloproteinase-1, and stimulated the STAT-1 pathway in cocultured endothelial cells. We used neutralizing Abs to show that the IFN-inducible protein 10 (CXCL10) and monokine-induced by IFN-gamma (CXCL9) chemokines produced by both PBMC and endothelial cells are pivotal in inducing these effects. Altogether these results suggest the existence of an IL-12-regulated circuit between endothelium and lymphocytes resulting in a shift of proteolytic homeostasis at site of tissue injury.