Nitric oxide modulates the expression of endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO^•). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO^• to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration.Effects of NO^•, either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO^• down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO^• was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression.In conclusion, during angiogenesis and leukocyte recruitment, NO^• regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO^•, reflecting the functions of the tissue they originate from.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Novel functions of vimentin in cell adhesion, migration, and signaling

Vimentin is the major intermediate filament (IF) protein of mesenchymal cells. It shows dynamically altered expression patterns during different developmental stages and high sequence homology throughout all vertebrates, suggesting that the protein is physiologically important. Still, until recently, the real tasks of vimentin have been elusive, primarily because the vimentin-deficient mice were originally characterized as having a very mild phenotype. Recent studies have revealed several key functions for vimentin that were not obvious at first sight. Vimentin emerges as an organizer of a number of critical proteins involved in attachment, migration, and cell signaling. The highly dynamic and complex phosphorylation of vimentin seems to be a likely regulator mechanism for these functions. The implicated novel vimentin functions have broad ramifications into many different aspects of cell physiology, cellular interactions, and organ homeostasis.     

A two‐hybrid screen identifies an unconventional role for the intermediate filament peripherin in regulating the subcellular distribution of the SNAP25‐interacting protein,SIP30

Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin up‐regulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious, and disease conditions remains unknown, but may be related to differential expression of spliced isoforms. To better understand peripherin function, we performed a yeast two‐hybrid screen on a mouse brain cDNA library using an assembly incompetent peripherin isoform, Per‐61, as bait. We identified new peripherin interactors with roles in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism. We focused on the interaction of Per‐61 and the constitutive isoform, Per‐58, with SNAP25 interacting protein 30 (SIP30), a neuronal protein involved in SNAP receptor‐dependent exocytosis. We found that peripherin and SIP30 interacted through coiled‐coil domains and colocalized in cytoplasmic aggregates in SW13vim(?) cells. Interestingly, Per‐61 and Per‐58 differentially altered the subcellular distribution of SIP30 and SNAP25 in primary motor neurons. Our findings suggest a novel role of peripherin in vesicle trafficking.

     

Plaque hemorrhage in carotid artery disease: Pathogenesis,clinical and biomechanical considerations

Stroke remains the most prevalent disabling illness today, with internal carotid artery luminal stenosis due to atheroma formation responsible for the majority of ischemic cerebrovascular events. Severity of luminal stenosis continues to dictate both patient risk stratification and the likelihood of surgical intervention. But there is growing evidence to suggest that plaque morphology may help improve pre-existing risk stratification criteria. Plaque components such a fibrous tissue, lipid rich necrotic core and calcium have been well investigated but plaque hemorrhage (PH) has been somewhat overlooked. In this review we discuss the pathogenesis of PH, its role in dictating plaque vulnerability, PH imaging techniques, marterial properties of atherosclerotic tissues, in particular, those obtained based on in vivo measurements and effect of PH in modulating local biomechanics.     

Use of in silico tools for classification of novel missense mutations identified in dystrophin gene in developing countries

DMD gene which is composed of 79 exons is the largest known gene located on X chromosome (Xp21). Point mutations in the dystrophin gene are responsible for 30–35% of cases with DMD/BMD. Mutation analysis of all the exons of the DMD gene is costly in developing countries, therefore, a few of the exons are selected to be analyzed routinely in clinical laboratories. In this study, direct sequencing was used for detection of point mutations in 10 exons of dystrophin gene in patients affected with DMD without detectable large rearrangements. Freely available programs were used to predict the damaging effects of the mutations. Point mutations were successfully detected in three patients. Three novel mutations, two missense mutations located on nonconservative domains and a single nucleotide deletion, were detected. Missense mutations were predicted to change splicing efficiency. Detection of point mutations by DNA analysis followed by prediction of the pathogenecity by using bioinformatic tool might be an asset to provide proper diagnosis or genetic counseling to patients and their family.     

人SNAP25基因重组腺病毒的构建与表达

利用RT-PCR法,从人胰腺组织扩增出SNAP25基因,定向克隆至pENTR-TOPO载体获得重组质粒pENTR/D-TOPO-SNAP25。经PCR及测序验证其阅读框正确。再与腺病毒载体pAD／CMV／V5一DEST 发生LR重组反应,SNAP25取代pAD／CMV／V5一DESTTM中的ccdB-CmR基因,获得重组腺病毒载体pAD／CMV／V5一DEST-SNAP25(pAD- SNAP25)。重组腺病毒载体经Pac I酶切,脂质体法转染HEK-293A细胞．以鼠抗人SNAP25单克隆抗体为一抗,做荧光及Western Blot检测,结果表明重组腺病毒pAD-SNAP25在HEK-293A细胞中包装成功。重组腺病毒pAD-SNAP25感染大鼠胰岛,结果表明在高糖浓度下,外源性SNAP25蛋白的表达促进了大鼠胰岛素分泌。     

Antiangiogenesis efficacy of nitric oxide donors

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis‐mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis‐mediated processes using the NO donor, S‐nitroso N‐acetyl penicillamine (SNAP) and S‐nitroso N‐acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)‐stimulated tube formation and serum‐induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85–95% inhibition) of cytokine (FGF2)‐induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2‐induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 μg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases. J. Cell. Biochem. 80:104–114, 2000. © 2000 Wiley‐Liss, Inc.     

Inhibition of endothelial cell proliferation and bFGF-induced phenotypic modulation by nitric oxide

S-nitroso-N-acetyl-D,L-acetylpenicillamine (SNAP), a chemical donor of NO, inhibited serum- and basic fibroblast growth factor (bFGF)-stimulated cultured endothelial cell (EC) proliferation in a dose-dependent manner. The inhibitory effect of NO was reversible after washoff of SNAP-containing media. Measurement of nitrate and nitrite in the media of SNAP-treated EC indicated that decomposition of SNAP into NO reached a stable level at or before 24 h; proliferation of EC was significantly inhibited for another 48 h and recovered thereafter if no additional SNAP was added. The level of NO produced by inhibitory concentrations of SNAP was comparable to NO levels produced by the induction of inducible nitric oxide synthase (iNOS) in smooth muscle cells or retinal pigmented epithelial cells. The growth-inhibitory effect of NO was unlikely to be due to cytotoxicity since 1) cells never completely lost their proliferative capacity even after 10 days of exposure to repeated additions of SNAP, 2) the inhibitory effect was reversible upon removal of NO and with the passage of time, and 3) NO did not reduce the number of cells that were growth-arrested with TGF-β1. In addition to its mitogenic effect, bFGF induced pronounced phenotypic changes, including suppression of contact inhibition, altered cell morphology, and scattering of the cells, in BPAEC cultures, whereas cells treated simultaneously with bFGF and NO did not exhibit these changes. These observations suggest that NO contributes to the regulation of angiogenesis and reendothelialization, processes that require EC proliferation, migration, and differentiation. © 1996 Wiley-Liss, Inc.