Reduction of obesity-associated white adipose tissue inflammation by rosiglitazone is associated with reduced non-alcoholic fatty liver disease in LDLr-deficient mice.

Obesity is associated with chronic low-grade inflammation that drives the development of metabolic diseases, including non-alcoholic fatty liver disease (NAFLD). We recently showed that white adipose tissue (WAT) constitutes an important source of inflammatory factors. Hence, interventions that attenuate WAT inflammation may reduce NAFLD development. Male LDLr-/- mice were fed a high-fat diet (HFD) for 9 weeks followed by 7 weeks of HFD with or without rosiglitazone. Effects on WAT inflammation and NAFLD development were analyzed using biochemical and (immuno)histochemical techniques, combined with gene expression analyses. Nine weeks of HFD feeding induced obesity and WAT inflammation, which progressed gradually until the end of the study. Rosiglitazone fully blocked progression of WAT inflammation and activated PPARγ significantly in WAT. Rosiglitazone intervention did not activate PPARγ in liver, but improved liver histology and counteracted the expression of genes associated with severe NAFLD in humans. Rosiglitazone reduced expression of pro-inflammatory factors in WAT (TNF-α, leptin) and increased expression of adiponectin, which was reflected in plasma. Furthermore, rosiglitazone lowered circulating levels of pro-inflammatory saturated fatty acids. Together, these observations provide a rationale for the observed indirect hepatoprotective effects and suggest that WAT represents a promising therapeutic target for the treatment of obesity-associated NAFLD.

The influence of respiration on criteria for transabdominal duplex examination of the splanchnic arteries in patients with suspected chronic splanchnic ischemia.

BACKGROUND: Duplex ultrasound imaging of the mesenteric vessels is often used as a first diagnostic tool to evaluate the mesenteric circulation in patients with unexplained chronic abdominal symptoms. Several studies on duplex criteria have been published; however, most studies are small and included not exclusively patients with symptoms suggestive of chronic mesenteric syndrome (CMS). This study evaluated the contribution of respiration-monitored duplex ultrasound imaging in the diagnosis of stenosis or occlusion of the mesenteric arteries in patients suspected of CMS and thereby improves the definition of the criteria for stenosis. METHODS: Between 1999 and 2007, 779 consecutive patients presented to our tertiary referral center for evaluation and treatment of CMS. Mesenteric artery duplex ultrasound imaging and angiography of the abdominal aorta and its branches were performed in 324 patients. Angiography was considered the gold standard for verifying the presence or absence of arterial pathology. Results from duplex imaging and angiography were compared to determine the optimal duplex criteria for stenosis. In addition, the contribution of expiration and inspiration on duplex imaging and angiography were established. RESULTS: Significantly higher peak systolic and end-diastolic velocities were found in the celiac artery (CA) and superior mesenteric artery (SMA) during expiration than during inspiration. Receiver operating characteristic curve analyses found respiration-dependent cutoff values for CA and SMA stenosis. The values corresponding with the highest accuracy (minimal false-negative and false-positive results) were determined. Peak systolic velocities cutoff points during expiration and inspiration were 280 and 272 cm/s, respectively, for the CA and 268 and 205 cm/s for the SMA. The end-diastolic velocity cutoff points during expiration and inspiration were 57 and 84 cm/s, respectively, for the CA and 101 and 52 cm/s for the SMA. Sensitivity for different duplex parameters in detecting mesenteric stenosis was 66% to 78% and specificity was 77% to 86%. CONCLUSIONS: This study proposes new criteria related to respiration for duplex ultrasound imaging of the mesenteric arteries in patients with symptoms suggestive of CMS. It emphasizes the importance of taking into account the effect of respiration on duplex parameters. The lower sensitivity and specificity in our study compared with other studies puts into perspective the position of duplex imaging in the work-up of patients with suspected CMS. Duplex results should be used as a guide, with a low threshold giving a higher negative predictive value and, consequently, a lower positive predictive value.

A clinical evaluation of statin pleiotropy: statins selectively and dose-dependently reduce vascular inflammation.

Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFκB regulated mediators (i.e. IL-6 (P<0.001) and MCP-1 (P<0.001)); shifted macrophage polarization towards a M2 phenotype (P<0.0003); selectively reduced macrophage-related markers such as cathepsin K and S (P<0.009 and 0.0027 respectively), and ALOX5 (P<0.0009), and reduced vascular wall NFκB activity (40 mg/day group, P<0.016). No effect was found on other cell types. Evaluation of the clinical efficacy of statins to reduce AAA progression did not indicate an effect of statins on aneurysm growth (P<0.337). Hence, in the context of AAA the clinical relevance of statins pleiotropy appears minimal.

Doxycycline for stabilization of abdominal aortic aneurysms: a randomized trial.

BACKGROUND: Doxycycline inhibits formation and progression of abdominal aortic aneurysms (AAAs) in preclinical models of the disease, but it is unclear whether and how this observation translates to humans. OBJECTIVE: To test whether doxycycline inhibits AAA progression in humans. DESIGN: Randomized, placebo-controlled, double-blind trial. (Dutch Trial Registry: NTR 1345) SETTING: 14 Dutch hospitals. PATIENTS: 286 patients with small AAAs between October 2008 and June 2011. INTERVENTION: Daily dose of 100 mg of doxycycline (n = 144) or placebo (n = 142) for 18 months. MEASUREMENTS: The primary outcome measure was aneurysm growth at 18 months, as estimated by repeated single-observer ultrasonography. Secondary outcomes included growth at 6 and 12 months and the need for elective surgery. RESULTS: Mean aneurysm diameter (approximately 43 mm) and other baseline characteristics were similar in both groups. Doxycycline treatment was associated with increased aneurysm growth (4.1 mm in the doxycycline group vs. 3.3 mm in the placebo group at 18 months; difference, 0.8 mm [95% CI, 0.1 to 1.4 mm]; P = 0.016 mm). Twenty-one patients receiving doxycycline and 22 patients receiving placebo had elective surgical repair (Kaplan­Meier estimates were 16.1% for those receiving doxycycline and 16.5% for those receiving placebo; difference, -0.4% [CI, -9.3% to 8.5%]; P = 0.83). Time to repair was similar in the groups (P = 0.92). LIMITATIONS: This study focuses on patients with small AAAs. As such, whether the data can be extrapolated to larger AAAs (>55 mm) is unclear. The high number of elective repairs (n = 43) was unanticipated. Moreover, the study did not follow patients who withdrew because of an adverse effect. CONCLUSION: This trial found that 18 months of doxycycline therapy did not reduce aneurysm growth and did not influence the need for AAA repair or time to repair. PRIMARY FUNDING SOURCE: The Netherlands Organisation for Health Research and Development, and the NutsOhra Fund.

Activator protein-1 (AP-1) signalling in human atherosclerosis: results of a systematic evaluation and intervention study.

Animal studies implicate the AP-1 (activator protein-1) pro-inflammatory pathway as a promising target in the treatment of atherosclerotic disease. It is, however, unclear whether these observations apply to human atherosclerosis. Therefore we evaluated the profile of AP-1 activation through histological analysis and tested the potential benefit of AP-1 inhibition in a clinical trial. AP-1 activation was quantified by phospho-c-Jun nuclear translocation (immunohistochemistry) on a biobank of aortic wall samples from organ donors. The effect of AP-1 inhibition on vascular parameters was tested through a double blind placebo-controlled cross-over study of 28 days doxycycline or placebo in patients with symptomatic peripheral artery disease. Vascular function was assessed by brachial dilation as well as by plasma samples analysed for hs-CRP (high-sensitivity C-reactive protein), IL-6 (interleukin-6), IL-8, ICAM-1 (intercellular adhesion molecule-1), vWF (von Willebrand factor), MCP-1 (monocyte chemoattractant protein-1), PAI-1 (plasminogen activator inhibitor-1) and fibrinogen. Histological evaluation of human atherosclerosis showed minimal AP-1 activation in non-diseased arterial wall (i.e. vessel wall without any signs of atherosclerotic disease). A gradual increase of AP-1 activation was found in non-progressive and progressive phases of atherosclerosis respectively (P<0.044). No significant difference was found between progressive and vulnerable lesions. The expression of phospho-c-Jun diminished as the lesion stabilized (P<0.016) and does not significantly differ from the normal aortic wall (P<0.33). Evaluation of the doxycycline intervention only revealed a borderline-significant reduction of circulating hs-CRP levels (-0.51 µg/ml, P=0.05) and did not affect any of the other markers of systemic inflammation and vascular function. Our studies do not characterize AP-1 as a therapeutic target for progressive human atherosclerotic disease.

A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease.

AIMS: Matrix metalloproteinases (MMP) and plasminogen activator (PA)/plasmin-mediated proteolysis, especially at the cell surface, play important roles in matrix degeneration and smooth muscle cell migration, which largely contributes to vein graft failure. In this study, a novel hybrid protein was designed to inhibit both protease systems simultaneously. MMP and plasmin activity were inhibited at the cell surface by this hybrid protein, consisting of the receptor-binding amino-terminal fragment (ATF) of urokinase-type PA, linked to both the tissue inhibitor of metalloproteinases (TIMP-1) and bovine pancreas trypsin inhibitor (BPTI), a potent protease inhibitor. The effect of overexpression of this protein on vein graft disease was studied. METHODS AND RESULTS: A non-viral expression vector encoding the hybrid protein TIMP-1.ATF.BPTI was constructed and validated. Next, cultured segments of human veins were transfected with this vector. Expressing TIMP-1.ATF.BPTI in vein segments resulted in a mean 36 ± 14% reduction in neointima formation after 4 weeks. In vivo inhibition of vein graft disease by TIMP-1.ATF.BPTI is demonstrated in venous interpositions placed into carotid arteries of hypercholesterolaemic APOE*3Leiden mice. After 4 weeks, vein graft thickening was significantly inhibited in mice treated with the domains TIMP-1, ATF, or BPTI (36-49% reduction). In the TIMP-1.ATF.BPTI-treated mice, vein graft thickening was reduced by 67±4%, which was also significantly stronger when compared with the individual components. CONCLUSION: These data provide evidence that cell surface-bound inhibition of the PA and MMP system by the hybrid protein TIMP-1.ATF.BPTI, overexpressed in distant tissues after electroporation-mediated non-viral gene transfer, is a powerful approach to prevent vein graft disease.

The pathophysiology of abdominal aortic aneurysm growth: corresponding and discordant inflammatory and proteolytic processes in abdominal aortic and popliteal artery aneurysms.

OBJECTIVE: There is remarkable controversy over the processes driving abdominal aneurysm growth. The inherent limitations of animal and human studies hamper elucidation of the key inflammatory and proteolytic processes. Human data are largely derived from surgical specimens that typically reflect the final stages of the disease process and thus do not allow distinction between primary and secondary processes. Clear epidemiologic and genetic associations between abdominal aortic aneurysm (AAA) and popliteal artery aneurysms (PAA) suggest that that these two pathologies share common grounds. On this basis, we reasoned that information of corresponding and discordant processes in these aneurysms might provide critical clues on the processes that are crucial for aneurysm progression. METHODS: Messenger RNA (semi-quantitative real-time polymerase chain reaction) and protein analysis (enzyme-linked immunosorbent assay, multiplex, Western blotting), and histology were performed on aneurysm wall samples obtained during elective PAA and AAA repair. Nonaneurysmal aorta tissue from organ donors was included as reference. RESULTS: Messenger RNA and protein analysis showed that PAA and AAA are both characterized by a marked activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) proinflammatory transcription factors, and hyperexpression of interleukin (IL)-6 and IL-8. Discordant findings were found for other inflammatory markers such as interferon-gamma, interferon-inducible protein 10, tumor necrosis factor-alpha, monocyte chemotactic protein-1, and macrophage inflammatory protein 1alpha and beta, which were all lower in PAA. On the cellular level, both pathologies exhibited profuse infiltration of macrophages, neutrophils, and T-helper cells. Results for B cells, plasma cells, and cytotoxic T cells were discordant, with minimal infiltration of these cell types in PAA. Evaluation of protease expression and activation showed that both conditions are dominated by increased matrix metalloproteinase 8 and 9, and cathepsin K, L and S expression and activation. CONCLUSION: This explorative study characterizes degenerative aneurysmal disease general inflammatory conditions that are dominated by profound activation of the NF-kappaB and AP-1 pathways, hyperexpression of IL-6 and IL-8, and neutrophil involvement. Discordant findings for interferon gamma, cytotoxic T cells, B cells, and plasma cells challenge a critical role for these factors in the process of aneurysm growth. Pharmaceutic strategies targeting the common components in AAA and PAA may prove effective for the stabilization of AAA.

In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor.

BACKGROUND: Smooth muscle cell (SMC) migration and proliferation are important in the development of intimal hyperplasia, the major cause of vein graft failure. Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system are pivotal in extracellular matrix degradation and, by that, SMC migration. Previously, we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the receptor-binding amino terminal fragment (ATF) of urokinase, reduces SMC migration and neointima formation in an in vitro restenosis model using human saphenous vein cultures more efficiently than both protease systems separately. Because use of viral gene delivery is difficult in clinical application, this study used nonviral delivery of TIMP-1.ATF plasmid to reduce vein graft disease in a murine bypass model. Nonviral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery, such as immune responses and short-term expression. METHODS: Plasmids encoding ATF, TIMP-1, TIMP-1.ATF, or luciferase, as a control, were injected and electroporated in both calf muscles of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3Leiden) mice (n = 8). One day after electroporation, a venous interposition of a donor mouse was placed into the carotid artery of a recipient mouse. In this model, vein graft thickening develops with features of accelerated atherosclerosis. Vein grafts were harvested 4 weeks after electroporation and surgery, and histologic analysis of the vessel wall was performed. RESULTS: Electroporation-mediated overexpression of the plasmid vectors resulted in a prolonged expression of the transgenes and resulted in a significant reduction of vein graft thickening (ATF: 36% +/- 9%, TIMP-1: 49% +/- 5%, TIMP-1.ATF: 58% +/- 5%; P < .025). Although all constructs reduced vein graft thickening compared with the controls, the luminal area was best preserved in the TIMP-1.ATF-treated mice. CONCLUSION: Intramuscular electroporation of TIMP-1.ATF inhibits vein graft thickening in vein grafts in carotid arteries of hypercholesterolemic mice. Binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface enhances the inhibitory effect of TIMP-1 on vein graft remodeling in vitro as well as in vivo and may be an effective strategy to prevent vein graft disease.

Limited value of digital subtraction angiography in the evaluation of cell-based therapy in patients with limb ischemia.

Cell-based therapy has been proposed as a novel strategy for patients with severe peripheral arterial disease by stimulating vascular growth. In clinical studies of this therapy, the emphasis has been on demonstrating recovery of clinical parameters, rather than on evaluation of blood flow recovery. Angiography is still the gold standard for the assessment of lower leg arteries. Therefore, we studied the usefulness of angiography in the evaluation of cell-based therapy. Sixteen patients with critical leg ischemia (ischemic rest pain or ulcers), or persistent (>12 months) profound disabling claudication were unilaterally treated with autologous bone marrow-derived mononuclear cells. Pre- and 6 months post-treatment digital subtraction angiographies (DSA) were assessed and compared in a blinded fashion twice by a panel of seven vascular surgeons and interventional radiologists. Inter- and intraobserver variability on qualitative (poor/moderate/rich) and semi-quantitative (increase/no difference/decrease) assessment of collateral circulation were evaluated. Agreement was expressed inter- and intraclass correlation coefficients (CC). Inter- and intraobserver agreement was moderate for the qualitative grading of collateral extent (CC = 0.46 and 0.60, respectively). Agreement was moderate (inter-CC = 0.60) to good (intra-CC = 0.73) for comparing pre- and post-treatment DSA. Clinical response was based on limb salvage, pain-free walking distance, ankle-brachial pressure index and pain scores. No difference was observed in the extent of collateral circulation between pre- and post treatment DSA after separate analysis of clinical responding and non-responding patients (P = 0.92). DSA is not a suited modality for the evaluation of therapeutic angiogenesis.

Local lentiviral short hairpin RNA silencing of CCR2 inhibits vein graft thickening in hypercholesterolemic apolipoprotein E3-Leiden mice.

OBJECTIVE: Inflammatory responses to vascular injury are key events in vein graft disease and accelerated atherosclerosis, which may result in bypass failure. The monocyte chemoattractant protein-1 (MCP-1)/CC-chemokine receptor (CCR)-2 pathway is hypothesized to play a central role. A murine model for vein graft disease was used to study the effect of local application of lentiviral short hairpin RNA (shRNA) targeted against CCR2. METHODS: A venous interposition was placed into the carotid artery of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3-Leiden) mice to induce vein graft thickening with features of accelerated atherosclerosis. To demonstrate the efficacy of the lentiviral shRNA targeting murine CCR2 (shCCR2) in blocking vein graft disease in vivo, lentiviral shCCR2 or a control lentivirus was used to infect the vein graft locally (n = 8). RESULTS: Vascular CCR2 and MCP-1 messenger RNA expression levels were significantly upregulated during lesion progression in the vein graft. Infection of smooth muscle cells (SMCs) with a lentiviral shRNA targeting shCCR2 completely abolished MCP-1-induced SMC migration and inhibited SMC proliferation in vitro (n = 3 per group). Morphometric analysis of sections of grafts showed a significant 38% reduction in vein graft thickening in the shCCR2-treated mice 4 weeks after surgery (control, 0.42 +/- 0.05 mm(2); shCCR2, 0.26 +/- 0.03 mm(2); P = .007). CONCLUSION: Vascular CCR2 contributes to vein graft disease, and local application of shRNA against CCR2 to the vessel wall prevents vein graft thickening in hypercholesterolemic mice, suggesting that local overexpressing of shRNA using organ-targeted lentiviral gene delivery may be a promising therapeutic tool to improve vein graft disease in bypassed patients. CLINICAL RELEVANCE: Vein graft disease is an important clinical issue that results from an inflammatory response. The monocyte chemoattractant protein (MCP)-1/CC-chemokine receptor (CCR)-2 pathway plays a key role in the initiation and development of vein graft disease. This study demonstrates that perivascular overexpression of short hairpin RNA, targeted against CCR2, inhibits vein graft thickening. These data show that organ-targeted gene therapy against CCR2 in the vessel wall could be a promising therapeutic tool to improve vein graft patency in bypassed patients.

Fluoroscopic Roentgen stereophotogrammetric analysis (FRSA) to study three-dimensional stent graft dynamics.

We report the clinical feasibility of fluoroscopic Roentgen stereophotogrammetric analysis (FRSA), a validated method to quantify real time three-dimensional (3D) dynamic motion of stent grafts and the first clinical results after abdominal and thoracic endovascular repair (EVAR). Stent graft motion was measured at 30 (stereo) frames per second, during the cardiac cycle and in the patient after abdominal EVAR, due to respiratory action. Translational motions of the center of mass, diameter change, and rotational and axial motion could be measured. Quantification of 3D motion was not available until now. FRSA can provide crucial information on the forces exerted on stent grafts and will, therefore, provide essential information for improvements in stent graft design.

MIF deficiency reduces chronic inflammation in white adipose tissue and impairs the development of insulin resistance, glucose intolerance, and associated atherosclerotic disease.

Chronic inflammation in white adipose tissue (WAT) is positively associated with obesity, insulin resistance (IR) and the development of type 2 diabetes. The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is an essential, upstream component of the inflammatory cascade. This study examines whether MIF is required for the development of obesity, IR, glucose intolerance, and atherosclerosis in the LDL receptor-deficient (Ldlr(-/-)) mouse model of disease. Ldlr(-/-) mice develop IR and glucose intolerance within 15 weeks, whereas Mif(-/-)Ldlr(-/-) littermates are protected. MIF deficiency does not affect obesity and lipid risk factors but specifically reduces inflammation in WAT and liver, as reflected by lower plasma serum amyloid A and fibrinogen levels at baseline and under inflammatory conditions. Conversely, MIF stimulates the in vivo expression of human C-reactive protein, an inflammation marker and risk factor of IR and cardiovascular disease. In WAT, MIF deficiency reduces nuclear c-Jun levels and improves insulin sensitivity; MIF deficiency also reduces macrophage accumulation in WAT and blunts the expression of two proteins that regulate macrophage infiltration (intercellular adhesion molecule-1, CD44). Mechanistic parallels to WAT were observed in aorta, where the absence of MIF reduces monocyte adhesion, macrophage lesion content, and atherosclerotic lesion size. These data highlight the physiological importance of chronic inflammation in development of IR and atherosclerosis and suggest that MIF is a potential therapeutic target for reducing the inflammatory component of metabolic and cardiovascular disorders.

Clinical trial of doxycycline for matrix metalloproteinase-9 inhibition in patients with an abdominal aneurysm: doxycycline selectively depletes aortic wall neutrophils and cytotoxic T cells.

BACKGROUND: Doxycycline has been shown to effectively inhibit aneurysm formation in animal models of abdominal aortic aneurysm. Although this effect is ascribed to matrix metalloproteinase-9 inhibition, such an effect is unclear in human studies. We reevaluated the effect of doxycycline on aortic wall protease content in a clinical trial and found that doxycycline selectively reduces neutrophil-derived proteases. We thus hypothesized that doxycycline acts through an effect on vascular inflammation. METHODS AND RESULTS: Sixty patients scheduled for elective open aneurysmal repair were randomly assigned to 2 weeks of low-, medium-, or high-dose doxycycline (50, 100, or 300 mg/d, respectively) or no medication (control group). Aortic wall samples were collected at the time of operation, and the effect of doxycycline treatment on vascular inflammation was evaluated. Independently of its dose, doxycycline treatment resulted in a profound but selective suppression of aortic wall inflammation as reflected by a selective 72% reduction of the aortic wall neutrophils and a 95% reduction of the aortic wall cytotoxic T-cell content (median values; P<0.00003). Evaluation of major inflammatory pathways suggested that doxycycline treatment specifically quenched AP-1 and C/EBP proinflammatory transcription pathways (P<0.0158, NS) and reduced vascular interleukin-6 (P<0.00115), interleukin-8 (P<0.00246, NS), interleukin-13 (P<0.0184, NS), and granulocyte colony-stimulating factor (P<0.031, NS) protein levels. Doxycycline was well tolerated; there were no adverse effects. CONCLUSIONS: A brief period of doxycycline treatment has a profound but selective effect on vascular inflammation and reduces aortic wall neutrophil and cytotoxic T-cell content. Results of this study are relevant for pharmaceutical stabilization of the abdominal aneurysm and possibly for other inflammatory conditions that involve neutrophils and/or cytotoxic T cells.

Doxycycline therapy for abdominal aneurysm: Improved proteolytic balance through reduced neutrophil content.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is thought to play a central role in abdominal aortic aneurysm (AAA) initiation. Doxycycline, a tetracycline analogue, has direct MMP-9-inhibiting properties in vitro, and it effectively suppresses AAA development in rodents. Observed inhibition of AAA progression, and contradictory findings in human studies evaluating the effect of doxycycline therapy on aortic wall MMP-9, suggest that the effects of doxycycline extend beyond MMP-9 inhibition and that the effect may be dose-dependent. METHODS: This clinical trial evaluated the effect of 2 weeks of low- (50 mg/d), medium- (100 mg/d), or high-dose (300 mg/d) doxycycline vs no medication in four groups of 15 patients undergoing elective AAA repair. The effect of doxycycline treatment on MMP and cysteine proteases, and their respective inhibitors, was evaluated by quantitative polymerase chain reaction, Western blot analysis, immunocapture protease activity assays, and immunohistochemistry. RESULTS: Doxycycline was well tolerated and no participants dropped out. Doxycycline treatment reduced aortic wall MMP-3 and MMP-25 messenger RNA expression (P < .045 and P < .014, respectively), selectively suppressed neutrophil collagenase and gelatinase (MMP-8 and MMP-9) protein levels (P < .013 and <.004, respectively), and increased protein levels of the protease inhibitors tissue inhibitor of metalloproteinase 1 and cystatin C (P < .029). As for the apparent selective effect on neutrophil-associated proteases, we sought for a reducing effect on aortic wall neutrophil content that was indeed confirmed by immunohistochemical analysis that revealed a 75% reduction in aneurysm wall neutrophil content (P < .001). CONCLUSIONS: Independent of its dose, short-term preoperative doxycycline therapy improves the proteolytic balance in AAA, presumably through an effect on aortic wall neutrophil content. This study provides a rationale for doxycycline treatment in patients with an AAA as well as in other (vascular) conditions involving neutrophil influx such as Kawasaki disease and Behçet disease.

Education in vascular surgery: critical issues around the globe-training and qualification in vascular surgery in Europe.

In 1958, the Union Européene des Médecins Spécialistes (UEMS), or European Union (EU) of Medical Specialists the European Union, was founded by the professional organizations of medical specialists in Europe. Among the objectives of the UEMS are to promote the highest level of patient care in the EU and to promote the harmonization of high-quality training programs within the various specialities throughout the EU. Within the 38 Specialist Sections of the UEMS are the European Boards, which are the working groups of the Specialist Sections. In 2005 Vascular Surgery was recognized as a separate and independent Section, a monospecialty, within the UEMS. The efforts of the UEMS are directed at facilitating the free exchange of training and work of trainees and medical specialists between EU countries. This situation, in combination with large differences in requirements and length of training in vascular surgery within the EU, stresses the importance of harmonization in training and certification in vascular surgery within the EU. For that reason, the European Board of Vascular Surgery has organized voluntary examinations yearly since 1996. The candidates who pass qualify as "Fellow of the European Board of Vascular Surgery" (FEBVS) since 2005. The first part of the examination evaluates the eligibility of the candidate (Certificate of Completion of Specialist Training, training center, logbook). The second part is a viva voce assessment that includes (1) case analyses, (2) a review of a scientific article, (3) an overall assessment, (4) a technical skills, and (5) an endovascular skills assessment. To pass the examination, the candidates must achieve a 67% success rate in each part of the examination. During the last 10 years, approximately 75% of the candidates have successfully taken the examination. In the near future the Section and Board, in close collaboration with the vascular societies in the EU, will develop a European vascular surgical syllabus and curriculum that will further harmonize and professionalize the training and certification of vascular surgery in Europe.

The impact of individualized evidence-based decision support on aneurysm patients' decision making, ideals of autonomy, and quality of life.

BACKGROUND: Patients with an asymptomatic abdominal aneurysm and their surgeon were randomized to receive a general brochure (GB) or an IB presenting survival information and a ranking of the treatment strategies. Before and after receiving the brochure, patients filled out questionnaires on their behavior during the consultation, ideals of patient autonomy, and quality of life. Surgeons answered a short checklist evaluating the consultation. RESULTS: One hundred patients participated, 49 in the intervention, 51 in the control group. The IB group had a better understanding of important issues in the treatment decision, had prepared more questions, and was less satisfied with the duration of the consultation. Their impression that the surgeon perceived them more as a medical problem than a patient with a problem increased. They agreed less with the surgeon's advice and lost some of their belief in "the doctor knows best.'' Beforehand, the IB group had a stronger preference for patient-based decisions, but afterward they displayed more surgeon-based decisions. No effects were seen on patients' quality of life. CONCLUSIONS: Individualized evidence-based information stimulated patients' active involvement but in the context of our study led to less patient-based decisions. Patient-made decisions and patient autonomy should, however, not be equated.

Enhanced expression and activation of pro-inflammatory transcription factors distinguish aneurysmal from atherosclerotic aorta: IL-6- and IL-8-dominated inflammatory responses prevail in the human aneurysm.

Inflammation plays a key role in the pathogenesis of an AAA (abdominal aortic aneurysm); however, the nature of the inflammatory factors and cellular response(s) involved in AAA growth is controversial. In the present study, we set out to determine the aortic levels of inflammatory cytokines in relation to downstream inflammatory transcription factors and cellular responses. A comparison of AAA wall samples with atherosclerotic wall samples taken from the same aortic region allowed AAA-specific inflammatory parameters to be identified that distinguish AAAs from ASD (aortic atherosclerotic disease). RT-PCR (real-time PCR), ELISA, Western blotting and immunohistochemistry were combined to assess cytokines and transcription factors at the mRNA and protein level, and their activation status. Compared with ASD, inflammatory parameters associated with Th1-type [T-bet, IL (interleukin)-2, IFN-gamma (interferon-gamma), TNF-alpha (tumour necrosis factor-alpha), IL-1alpha and cytotoxic T-cells] and Th2-type [GATA3, IL-4, IL-10, IL-13 and B-cells] responses were all increased in AAA samples. Evaluation of major downstream inflammatory transcription factors revealed higher baseline levels of C/EBP (CCAAT/enhancer-binding protein) alpha, beta and delta in the AAA samples. Baseline p65 NF-kappaB (nuclear factor kappaB) and c-Jun [AP-1 (activator protein-1)] levels were comparable, but their activated forms were strongly increased in the AAA samples. Downstream target genes of p65 NF-kappaB, c-Jun, IL-6 and IL-8 were hyperexpressed. Molecular and cellular processes associated with IL-6 and IL-8 hyperactivation were enhanced in the AAA samples, i.e. the expression of phospho-STAT-3 (signal transducer and activator of transcription-3) and perforin were elevated, and the content of plasma cells, neutrophils and vasa vasorum was increased. In conclusion, our findings demonstrate that an AAA is a general inflammatory condition which is characterized by enhanced expression and activation of pro-inflammatory transcription factors, accompanied by IL-6 and IL-8 hyperexpression and exaggerated downstream cellular responses, which together clearly distinguish an AAA from ASD.

Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.

For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a plasmid in which two opposing human polymerase III promoters (H1 and U6) drive the expression of siRNA constructs that form functional double-stranded siRNAs, in combination with in vivo bioluminescence imaging, we were able to knock down exogenous delivered luciferase for at least 100 days in murine calf muscles. This effect was sequence specific, because scrambled siRNA had no effect. Moreover, we were able to demonstrate in vivo reduction of endogenous TLR4 expression for at least 1 week, using a similar vector expressing an siRNA for TLR4 in the muscle. In this study, we demonstrate that in vivo suppression of both endogenous (for at least 1 week) and introduced genes (>100 days) is feasible via plasmid-driven siRNA expression after electroporation-mediated intramuscular gene transfer. With this approach the short-term effect of oligonucleotides and the drawbacks of viral gene delivery, like immunological responses, could be circumvented. Therefore, this application of RNA interference is a useful tool with which to investigate gene function and might be promising as a therapeutic tool for locally acting diseases such as restenosis or tumors.

Expression of vascular endothelial growth factor, stromal cell-derived factor-1, and CXCR4 in human limb muscle with acute and chronic ischemia.

OBJECTIVE: Vascular endothelial growth factor (VEGF)-induced stromal cell-derived factor-1 (SDF-1) has been implicated in angiogenesis in ischemic tissues by recruitment of CXCR4-positive bone marrow-derived circulating cells with paracrine functions in preclinical models. Here, evidence for this is provided in patients with peripheral artery disease. METHODS AND RESULTS: Expression patterns of VEGF, SDF-1, and CXCR4 were studied in amputated limbs of 16 patients. VEGF-A was expressed in vascular structures and myofibers. SDF-1 was expressed in endothelial and subendothelial cells, whereas CXCR4 was expressed in proximity to capillaries. VEGF-A, SDF-1, and CXCR4 expressions were generally decreased in ischemic muscle as compared with nonischemic muscle in patients with chronic ischemia (0.41-fold, 0.97-fold, and 0.54-fold induction [medians], respectively), whereas substantially increased in 2 patients with acute-on-chronic ischemia (3.5- to 65.8-fold, 3.9- to 19.0-fold, and 4.1- to 30.6-fold induction, respectively). Furthermore, these gene expressions strongly correlated with capillary area. Only acute ischemic tissue displayed a high percentage of hypoxia-inducible factor-1alpha-positive nuclei. CONCLUSIONS: These data suggest that VEGF and SDF-1 function as pro-angiogenic factors in patients with ischemic disease by perivascular retention of CXCR4-positive cells. Furthermore, these genes are downregulated in chronic ischemia as opposed to upregulated in more acute ischemia. The VEGF-SDF-1-CXCR4 pathway is a promising target to treat chronic ischemic disease.