摘要:

In human occluded saphenous vein grafts, we previously demonstrated cytotoxic foam cells, presumably derived from macrophages engulfing platelets. In the present study, we investigated whether platelet phagocytosis occurs in human atherosclerotic plaques, whether this activates macrophages, and whether the platelet constituent, amyloid precursor protein (APP), was involved. Immunohistochemistry documented the presence of APP, &bgr;-amyloid peptide (A&bgr;, cleaved from APP), and platelets (CD9), along with inducible NO synthase (iNOS) and cyclooxygenase-2, two markers of macrophage activation, around microvessels in advanced human carotid artery plaques (n=18). A&bgr; colocalized with iNOS-expressing macrophages that were often surrounded by platelets. In vitro, murine J774 and human THP-1 macrophages were incubated with or without washed human platelets. Coincubation of macrophages and platelets led to platelet phagocytosis (electron and confocal microscopy) and formation of lipid-, APP-, and A&bgr;-containing foam cells. These expressed iNOS mRNA (reverse transcription–polymerase chain reaction) and protein and produced nitrite and tumor necrosis factor-&agr; (ELISA). Macrophage pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, a protease inhibitor, reduced APP processing and inhibited NO biosynthesis induced by platelet phagocytosis but not by lipopolysaccharides. Human atherosclerotic plaques and J774 and THP-1 macrophages contained mRNA of the APP-cleaving enzyme &bgr;-secretase. This is the first demonstration of A&bgr;, a peptide extensively studied in Alzheimer’s disease, in human atherosclerotic plaques. It was present in activated iNOS-expressing perivascular macrophages that had phagocytized platelets. In vitro studies indicate that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to A&bgr;, resulting in iNOS induction. This represents a novel mechanism for macrophage activation in atherosclerosis.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)–regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase–polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II–mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Neutrophil-induced coronary microvascular leakage represents an important pathophysiological consequence of ischemic and inflammatory heart diseases. The precise mechanism by which neutrophils regulate endothelial barrier function remains to be established. The aim of this study was to examine the microvascular endothelial response to neutrophil activation with a focus on myosin light chain kinase (MLCK)-mediated myosin light chain (MLC) phosphorylation, a regulatory process that controls cell contraction. The apparent permeability coefficient of albumin (Pa) was measured in intact isolated porcine coronary venules. Incubation of the vessels with C5a-activated neutrophils induced a time- and concentration-dependent increase in Pa. The hyperpermeability response was significantly attenuated during inhibition of endothelial MLC phosphorylation with the selective MLCK inhibitor ML-7 and transfection of a specific MLCK-inhibiting peptide. In contrast, transfection of constitutively active MLCK elevated Pa, which was abolished by ML-7. In addition to the vessel study, albumin transendothelial flux was measured in cultured bovine coronary venular endothelial monolayers, which displayed a hyperpermeability response to neutrophils and MLCK in a pattern similar to that in venules. Importantly, neutrophil stimulation caused MLC phosphorylation in endothelial cells in a time course closely correlated with that of the hyperpermeability response. Consistently, the MLCK inhibitors abolished neutrophil-induced MLC phosphorylation. Furthermore, immunohistochemical observation of neutrophil-stimulated endothelial cells revealed an increased staining for phosphorylated MLC in association with contractile stress fiber formation and intercellular gap development. Taken together, the results suggest that endothelial MLCK activation and MLC phosphorylation play an important role in mediating endothelial barrier dysfunction during neutrophil activation.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Insulin-like growth factor (IGF)-1 and the type I IGF-1 receptor are important regulators of vascular function that may contribute to cardiovascular disease. We hypothesized that IGF-1 causes endothelial cell dysfunction and expression of neutrophil and monocyte adhesion molecules by enhancing pro-inflammatory cytokine signal transduction. Long-term IGF-1 treatment of endothelial cells potentiated c-Jun and nuclear factor NF-&kgr;B activation by tumor necrosis factor (TNF)-&agr; and enhanced TNF-&agr;–mediated adhesion molecule expression. In response to IGF-1 treatment, the expression of kinases in the c-Jun/c-Jun NH2-terminal kinase signaling pathway (MEKK1, MEK4, and JNK1/2) was unchanged, but expressions of insulin receptor substrate-1 and Grb2-associated binder-1 (Gab1) were significantly decreased. Because Gab1 is involved in both c-Jun and NF-&kgr;B activation by TNF-&agr;, we focused on Gab1-dependent signaling. Gab1 inhibited c-Jun and NF-&kgr;B transcriptional activation by TNF-&agr;. Interestingly, Gab1 inhibited c-Jun transcriptional activity induced by MEKK3 but not MEKK1 and MEK4. Gab1 associated with MEKK3, and a catalytically inactive form of MEKK3 inhibited TNF-&agr;–induced c-Jun and NF-&kgr;B transcriptional activation, suggesting a critical role for Gab1 and MEKK3 in TNF-&agr; signaling. These data demonstrate that Gab1 and MEKK3 play important roles in endothelial cell inflammation via regulating the activation of c-Jun and NF-&kgr;B. Furthermore, the IGF-1–mediated downregulation of Gab1 expression represents a novel mechanism to promote vascular inflammation and atherosclerosis.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID