Outcome of giant cell arteritis of the arm arteries managed with medical treatment alone: cross-sectional follow-up study.

OBJECTIVE: To assess the long-term outcome of GCA of the arm arteries under medical treatment alone. METHODS: Retrospective cross-sectional study of 34 patients with a diagnosis of GCA involving the arm arteries (i.e. subclavian, axillary and/or brachial arteries). All patients were managed with immunosuppressive treatment and antiplatelet agents and followed clinically and with colour duplex sonography. Characteristics of patients with and without relief of ischaemic upper extremity symptoms during follow-up were compared using Fisher's exact test and the Mann-Whitney U-test. RESULTS: Sixteen of 34 patients (47.1%) suffered from arm claudication at the time of diagnosis (bilateral symptoms in 8 patients). During a mean follow-up of 21.9 (17.1) months, none of the patients developed new ischaemic symptoms of the upper extremities, and five patients became asymptomatic. Critical limb ischaemia did not occur. Symptom relief was significantly less frequent in patients with symptomatic ischaemia of the right arm vs the left arm and was negatively associated with the presence of anaemia and subclavian artery involvement (all P < 0.05). In 32.4% of patients, the vasculitic wall thickening, as depicted by colour duplex sonography, completely disappeared. CONCLUSION: With medical treatment alone, the prognosis of GCA of the arm arteries is benign. Patients with unilateral left-sided ischaemic symptoms may have a higher probability of complete symptom relief than patients with right-sided or bilateral arm claudication.

eNOS protects from atherosclerosis despite relevant superoxide production by the enzyme in apoE mice.

BACKGROUND: All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme. PRINCIPAL FINDINGS: Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE(-/-)/eNOS(-/-)), while P/E-interactions did not differ, compared to apoE(-/-). eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE(-/-) vessels. CONCLUSION: Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE(-/-)/eNOS(-/-) vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE(-/-) atherosclerosis but does not negate the enzyme's strong protective effects.

Oxidative stress and compartment of gene expression determine proatherosclerotic effects of inducible nitric oxide synthase.

Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2(-)), or both; these radicals may then react to form peroxynitrite. Alternatively, O2(-) radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE(-/-)) and apoE/iNOS double knockout (apoE(-/-)/ iNOS(-/-)) mice to assess vascular NO, O2(-), and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2(-), leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression.

Expression of neuronal nitric oxide synthase splice variants in atherosclerotic plaques of apoE knockout mice.

OBJECTIVE: We previously reported that deletion of brain type neuronal nitric oxide synthase-alpha (nNOS-alpha) accelerates atherosclerosis in apolipoproteinE (apoE) knockout (ko) mice. The regulation of nNOS expression is complex, involving the generation of mRNA splice variants. The current study investigates occurrence and distribution of nNOS variants in atherosclerotic lesions of apoE ko and apoE/nNOS-alpha double ko (dko) animals. METHODS: Mice were fed a high fat diet for 20 weeks. Immunohistochemistry and western blot analysis were performed using antibodies detecting the carboxy terminal-, or amino terminal-residue of the nNOS protein. Confocal microscopy and in situ hybridization were used to identify the compartment of cellular expression. RESULTS: In situ hybridization revealed the presence of nNOS-alpha and -gamma mRNA variants in apoE ko plaques, while only nNOS-gamma was detectable in apoE/nNOS dko plaques. Consistent with mRNA expression nNOS-alpha protein can be detected in the neointima of apoE ko, but not apoE/nNOS dko animals. In contrast, the carboxy terminal antibody stained the neointima and media in apoE ko vessels and showed residual nNOS immunoreactivity in apoE/nNOS dko lesions. Confocal microscopy showed predominant nNOS expression in vascular smooth muscle cells, while colocalization with macrophages was less pronounced. CONCLUSIONS: Our study shows that nNOS-alpha and -gamma splice variants are expressed in atherosclerotic plaques of apoE ko mice. nNOS variants colocalized with markers for vascular smooth muscle cells and macrophages but not for endothelial cells. Since nNOS-alpha is atheroprotective, other nNOS splice variants which differ in enzyme kinetic and subcellular localization may also influence plaque formation.

Ezetimibe potently reduces vascular inflammation and arteriosclerosis in eNOS-deficient ApoE ko mice.

OBJECTIVE: Hypercholesterolemia is associated with decreased vascular nitric oxide bioavailability and deletion of endothelial nitric oxide synthase (eNOS) markedly accelerates atherosclerosis development in apolipoprotein E knockout (apoE ko) mice. The current study tests whether atheroprotection provided by a lipid lowering therapy with Ezetimibe depends on eNOS. METHODS/RESULTS: ApoE ko and apoE/eNOS double ko (dko) mice received a high fat diet with or without 0.05% Ezetimibe. Ezetimibe significantly reduced plasma cholesterol concentrations and atherogenic lipoproteins in both genotypes to a similar extent. Moreover, the drug reduced vascular inflammation, as it significantly reduced vascular cell adhesion molecule-1 (VCAM-1) expression and vascular CD14 expression, a marker for mononuclear cell infiltration, in both genotypes. Neither NOS protein expression nor vascular reactivity of aortic rings was changed in apoE ko mice following Ezetimibe treatment. Significant lesion reduction was seen in Ezetimibe-treated male and female apoE ko and apoE/eNOS dko animals (p

Overlapping and distinct roles for PI3Kbeta and gamma isoforms in S1P-induced migration of human and mouse endothelial cells.

AIMS: Sphingosine-1-phosphate (S1P), a key regulator of vascular homeostasis and angiogenesis, promotes endothelial cell migration via stimulation of phosphoinositide 3-kinase (PI3K). The aim of this study was to identify the role of PI3Kbeta and gamma isoforms and their downstream effector pathways in mediating endothelial cell migration induced by S1P. METHODS AND RESULTS: Experiments were performed in human umbilical vein endothelial cells (HUVEC) and murine lung endothelial cells (MLEC). A combination of specific inhibitors, RNA interference, and PI3Kgamma(-/-) mice were used to investigate the role of PI3Kbeta and gamma isoforms in endothelial cell migration. Both PI3Kbeta and gamma isoforms are required for full migration induced by S1P, with Rac1 being a major mediator downstream of both isoforms. In addition, PI3Kbeta but not PI3Kgamma mediates migration via Akt but independent of Rac1 and endothelial NO synthase (eNOS). Further, a S1P-mediated activation of extracellular signal-regulated kinases (Erk) 1/2 contributes to the chemotactic response independent of PI3Kbeta or PI3Kgamma. CONCLUSIONS: Our data demonstrate that both PI3Kbeta and PI3Kgamma isoforms are required for S1P-induced endothelial cell migration through activation of Rac1. In addition, PI3Kbeta initiates an Akt-sensitive chemotactic response which is independent of Rac1 and eNOS. Thus, PI3Kbeta and PI3Kgamma have both overlapping and distinct roles in regulating endothelial cell migration, which may underlie S1P-triggered angiogenic differentiation.

Large external iliac vein aneurysm in a patient with a post-traumatic femoral arteriovenous fistula.

We report about a young patient with a large aneurysm of the left external iliac vein associated with a traumatic arteriovenous fistula between the left superficial femoral artery and the femoral vein after a stab wound 20 years ago. The patient presented with swelling of the left leg, which developed during the past years and worsened after saphenectomy 12 months before hospital admission. The chronically hyperperfused common iliac artery proximal to the arteriovenous fistula was compressing the common iliac vein. The venous outflow obstruction and subsequent venous hypertension render a possible explanation for the formation of the iliac vein aneurysm. Surgical repair of the venous aneurysm by interposition grafting and closure of the arteriovenous fistula was successful. A postoperative computed tomography scan showed a 50% size reduction of the feeding artery, underlining the ability of the arterial system to normalize arterial diameter in response to flow reduction, even after a high flow situation had existed for probably >20 years.

Native C-reactive protein induces endothelial dysfunction in ApoE-/- mice: implications for iNOS and reactive oxygen species.

OBJECTIVE: In addition to being a risk marker for cardiovascular disease, recent data suggests that C-reactive protein (CRP) induces endothelial dysfunction and promotes oxidative stress. We evaluated the effects of two conformers of CRP (pentameric, or native [nCRP], versus monomeric, or modified [mCRP]) on vessel function and production of reactive oxygen species (ROS) in an in-vivo model of atherosclerosis. METHODS AND RESULTS: Female ApoE(-/-) mice, fed a "western-type" diet, were treated with either human nCRP or mCRP (2.5mg/kg s.c., weekly) or saline for 8 weeks. Endothelium-dependent and endothelium-independent vascular functions were assessed in isolated aortic rings under isometric conditions. Production of ROS in aortic rings was measured by electron spin resonance (ESR). Endothelium-dependent relaxation was impaired in nCRP-treated but not in mCRP-treated ApoE(-/-) mice. This impairment was reversed by preincubation with an inhibitor of inducible nitric oxide synthase (iNOS). Endothelium-independent relaxation, and iNOS and endothelial NOS (eNOS) protein expressions were similar among all groups. ESR experiments revealed lesser amounts of superoxide in the nCRP group as compared to the saline group, which is consistent with an increased transformation of NO to peroxynitrite. CONCLUSIONS: nCRP can facilitate cardiovascular disease through impairment of endothelium-dependent vasoreactivity, in a manner that involves increased iNOS activity and a potential for increased peroxynitrite formation.

Tissue-specific effects of the nuclear factor kappaB subunit p50 on myocardial ischemia-reperfusion injury.

Nuclear factor kappaB (NF-kappaB) is a ubiquitous transcription factor activated by various stimuli implicated in ischemia-reperfusion injury. However, the role of NF-kappaB in cardiac ischemia-reperfusion injury has not yet been well defined. Therefore, we investigated reperfusion damage in mice with targeted deletion of the NF-kappaB subunit p50. Electrophoretic mobility shift assays validated NF-kappaB activation in wild-type (WT) but not p50 knockout (KO) mice. KO and WT animals underwent 30 minutes of coronary artery ligation and 24 hours of reperfusion in vivo. Ischemia-reperfusion damage was significantly reduced in the p50 KO when compared with matching WT mice. Although adhesion molecules such as intercellular adhesion molecule were up-regulated in left ventricles of p50 KO animals, fewer neutrophils infiltrated the infarct area, suggesting leukocytes as a potential mediator of the protection observed in the p50 KO. This was confirmed in adoptive transfer experiments: whereas transplantation of KO bone marrow in KO animals sustained the protective effect on ischemia-reperfusion injury, transplantation of WT bone marrow in KO animals abolished it. Thus, deletion of the NF-kappaB subunit p50 reduces ischemia-reperfusion injury in vivo, associated with less neutrophil infiltration. Bone marrow transplantation experiments indicate that impaired NF-kappaB activation in p50 KO leukocytes attenuates cardiac damage.

Atheroprotective effects of neuronal nitric oxide synthase in apolipoprotein e knockout mice.

OBJECTIVE: All 3 isoforms of the nitric oxide synthase (NOS) are expressed in atherosclerotic lesions. To test whether neuronal NOS (nNOS) deficiency affects atherosclerosis, we studied apoE/nNOSalpha double knockout (DKO) and apolipoprotein E (apoE) knockout (KO) control mice. METHODS AND RESULTS: Lesion area was significantly increased in male DKO (66%) mice after 14 weeks and in female DKO animals (31%) after 24 weeks of "western" diet. Moreover, mean arterial blood pressure was significantly reduced in female DKO animals. Immunohistochemistry revealed nNOS expression in the neointima of KO mice. In DKO animals, residual nNOS staining was caused by the presence of nNOS splice variants. Whereas nNOSalpha was present in vessels of KO and absent in DKO animals, nNOSgamma was expressed in KO and DKO mice. CONCLUSIONS: nNOSalpha protects against atherosclerosis as nNOSalpha deletion leads to an increase in plaque formation in apoE/nNOSalpha DKO mice. Female DKO mice showed a significant reduction in mean arterial blood pressure. Additionally, we found expression of nNOS splice variants in vessels of apoE KO mice. Our data highlights nNOSalpha overexpression as a potential therapeutic strategy and naturally occurring splice variants that lack exon 2 of the nNOS gene as a potential risk factor for vascular disease.

Integrin-linked kinase regulates endothelial cell survival and vascular development.

Integrin-linked kinase (ILK) is a phosphoinositide 3-kinase-dependent serine/threonine kinase that interacts with beta integrins. Here we show that endothelial cell (EC)-specific deletion of ILK in mice confers placental insufficiency with decreased labyrinthine vascularization, yielding no viable offspring. Deletion of ILK in zebra fish using antisense morpholino oligonucleotides results in marked patterning abnormalities of the vasculature and is similarly lethal. To dissect potential mechanisms responsible for these phenotypes, we performed ex vivo deletion of ILK from purified EC of adult mice. We observed downregulation of the active-conformation of beta1 integrins with a striking increase in EC apoptosis associated with activation of caspase 9. There was also reduced phosphorylation of the ILK kinase substrate, Akt. However, phenotypic rescue of ILK-deficient EC by wild-type ILK, but not by a constitutively active mutant of Akt, suggests regulation of EC survival by ILK in an Akt-independent manner. Thus, endothelial ILK plays a critical role in vascular development through integrin-matrix interactions and EC survival. These data have important implications for both physiological and pathological angiogenesis.

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells.

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N(omega)-monomethyl-l-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation.