Human umbilical vein endothelial cells (HUVECs) have played a major role as a model system for the study of the regulation of endothelial cell function and the role of the endothelium in the response of the blood vessel wall to stretch, shear forces, and the development of atherosclerotic plaques and angiogenesis. Here, we use HUVECs and human microvascular endothelial cells to study the role of the HMG-CoA reductase inhibitor, simvastatin, and the small GTP-binding protein Rho in the regulation of angiogenesis. Simvastatin inhibited angiogenesis in response to FGF-2 in the corneal pocket assay of the mouse and in vascular endothelial growth factor (VEGF)-stimulated angiogenesis in the chick chorioallontoic membrane. Furthermore, simvastatin inhibited VEGF-stimulated tube formation by human dermal microvascular endothelial cells and the formation of honeycomb-like structures by HUVECs. The effect was dose-dependent and was not secondary to apoptosis. Geranylgeranyl-pyrophosphate (GGPP), a product of the cholesterol metabolic pathway that serves as a substrate for the posttranslational lipidation of RhoA, was required for membrane localization, but not farnesylpyrophosphate (FPP), the substrate for the lipidation of Ras. Furthermore, GGTI, a specific inhibitor of GGPP, mimicked the effect of simvastatin of tube formation and the formation of honeycombs whereas FTI, a specific inhibitor of the farnesylation of Ras, had no effect. Adenoviral expression of a DN-RhoA mutant mimicked the effect of simvastatin on tube formation and the formation of honeycombs, whereas a dominant activating mutant of RhoA reversed the effect of simvastatin on tube formation. Finally, simvastatin interfered with the membrane localization of RhoA with a dose-dependence similar to that for the inhibition of tube formation. Simvastatin also inhibited the VEGFstimulated phosphorylation of the VEGF receptor KDR, and the tyrosine kinase FAK, which plays a role in cell migration. These data demonstrate that simvastatin interfered with angiogenesis via the inhibition of RhoA. Data supporting a role for angiogenesis in the development and growth of atherosclerotic plaques suggest that this antiangiogenic effect of Statins might prevent the progression of atherosclerosis via the inhibition of plaque angiogenesis.

A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence. It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1 : 1 ratio of FAD to the polypeptide chain.The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H202. The development of enzyme-based biosensors has recently become a subject of particular interest (Scheller and Schubert, 1989; Cass, 1990), and enzymes whichcatalyze redox reactions were frequently used for this purpose. If the product or the substrate in this reaction is electroactive, their concentrations can be measured arnperometrically (Cass, 1990). Thus, the function of this type of biosensor is based on the measurement of oxygen consumption or the formation of hydrogen peroxide (Mosbach, 1988). Amperometric biosensors for substrates which are enzymatically coupled to NAD'/NADH have attracted considerable attention due to their clinical and biotechnological relevance.The flavoenzyme NADH oxidase (NADH :oxygen oxidoreductase) catalyzes the oxidation of NADH by the reduction of oxygen. The advantage of the application of NADH oxidase as an amperometric enzyme electrode lies not only in its sensitivity and specificity, but also in its efficient regeneration of NAD'.Several bacterial NADH oxidases have been isolated and characterized to date. The NADH oxidase derived from aerobically grown Streptococcus faecalis catalyzes the direct four-electron reduction of O 2 to 2 H 2 0 (Hoskins et aI., 1%2;Correspondence to

Objective-Abdominal aortic aneurysm (AAA) is a life-threatening disease affecting almost 10% of the population over age 65. Generation of AAAs by infusion of angiotensin (Ang) II in apolipoprotein E-knockout (ApoE Ϫ/Ϫ ) mice is an animal model which supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA. The effect of statins on AngII-mediated AAA formation and the associated neovascularization is not known. Here we determined the effect of simvastatin and the ERK inhibitor, CI1040, on AngII-stimulated AAA formation. Methods and Results-ApoEϪ/Ϫ mice infused for 28 days with AngII using osmotic minipumps were treated with placebo, 10 mg/kg/d simvastatin, or 100 mg/kg/d CI1040. 95% of AngII-treated mice developed AAA with neovascularization of the lesion, increased ERK phosphorylation, MCP-1 secretion, and MMP activity. These effects were markedly reversed by simvastatin and in part by CI1040. Furthermore, simvastatin and the ERK inhibitor U0126 reversed AngII-stimulated angiogenesis and MMP secretion by human umbilical vein endothelial cells. Key Words: angiotensin II Ⅲ abdominal aortic aneurysm Ⅲ statin Ⅲ ERK inhibition Ⅲ angiogenesis A bdominal aortic aneurysm (AAA) is a potentially lifethreatening degenerative vascular disease that affects 6% to 9% of men over the age of 65 years, claiming more than 15 000 lives annually. 1 Currently, surgical repair is the only effective method of AAA treatment. 2 Studies of the pathophysiology of AAA have demonstrated that human aneurysmal tissues are characterized by (1) chronic inflammation of the aortic wall with the accumulation of macrophages and MCP-1 secretion 3 ; (2) progressive degradation of extracellular matrix including elastin and collagen 4 ; (3) increased activity of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9 5 ; (4) reendothelialization of the dilated luminal surface of the vessel wall and pronounced neovascularization of the media and adventitia. 6,7 Several animal models of AAA have been developed in the mouse, including injury of the aortic wall with calcium chloride 8 or elastase. 9 Infusion of AngII via subcutaneous osmotic minipumps in ApoE Ϫ/Ϫ mice has also been shown to result in reproducible formation of suprarenal AAAs, which exhibit many characteristics of the human disease including secretion of MCP-1, 5 macrophage infiltration, secretion of MMPs, disruption of the media, rupture of the elastic layer, and neovascularization. 10 This is consistent with the finding that AngII not only plays a role in the control of cardiovascular and renal homeostasis but also affects vascular endothelial cell function, macrophage activation, 11 and the contraction, migration, and proliferation of vascular smooth muscle cells. 12 This model supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA and is thus an important model for the study of mechanisms of AAA formation. Conclusions-TheseThe mitogen-activated protein (MAP) kinase family, including ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK...

Abdominal Aortic Aneurysm (AAA) is a major cause of mortality and morbidity in men over 65 years of age. Male apolipoprotein E knockout (ApoE -/-) mice infused with angiotensin II (AngII) develop AAA. Although AngII stimulates both JAK/STAT and Tolllike receptor 4 (TLR4) signaling pathways, their involvement in AngII mediated AAA formation is unclear. Here we used the small molecule STAT3 inhibitor, S3I-201, the TLR4 inhibitor Eritoran and ApoE -/- TLR4-/-mice to evaluate the interaction between STAT3 and TLR4 signaling in AngII-induced AAA formation. ApoE -/-mice infused for 28 days with AngII developed AAAs and increased STAT3 activation and TLR4 expression. Moreover, AngII increased macrophage infiltration and the ratio of M1 (proinflammatory)/M2 (healing) macrophages in aneurysmal tissue as early as 7-10 days after AngII infusion. STAT3 inhibition with S3I-201 decreased the incidence and severity of AngII-induced AAA formation and decreased MMP activity and the ratio of M1/M2 macrophages. Furthermore, AngII-mediated AAA formation, MMP secretion, STAT3 phosphorylation and the ratio of M1/M2 macrophages were markedly decreased in ApoE -/-TLR4 -/-mice, and in Eritoran-treated ApoE -/-mice. TLR4 and pSTAT3 levels were also increased in human aneurysmal tissue. These data support a role of pSTAT3 in TLR4 dependent AAA formation and possible therapeutic roles for TLR4 and/or STAT3 inhibition in AAA.

As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.