Corey S. Moran

Association of Osteoprotegerin With Human Abdominal Aortic Aneurysm Progression

Background—Abdominal aortic aneurysm (AAA) is characterized by destruction of the arterial media associated with loss of vascular smooth muscle cells, infiltration of mononuclear cells, and high concentration of metalloproteinases (MMPs) and cytokines. Osteoprotegerin (OPG) has recently been identified in atherosclerosis. The presence and functional importance of OPG in human AAA was investigated. Methods and Results—In 146 men with small AAA followed up by ultrasound for 3 years, serum OPG was weakly correlated with aneurysm growth rate. Western analysis showed 3-, 8-, and 12-fold-greater OPG concentrations in human AAA biopsies compared with biopsies of atherosclerotic narrowed aorta (1.4±0.1 versus 0.5±0.1 ng/mg tissue; P=0.002), postmortem nondiseased abdominal aorta (1.4±0.1 versus 0.2±0.1 ng/mg tissue; P<0.001), and nondiseased thoracic aorta (1.4±0.1 versus 0.1±0.06 ng/mg tissue; P<0.001). Healthy human aortic vascular smooth muscle cells incubated with recombinant human (rh)OPG (0 to 20 ng rhOPG/105 cells per 1 mL per 24 hours) developed an aneurysmal phenotype defined by impaired cell proliferation (P<0.001), increased apoptosis (P<0.01), and increased MMP-9 (92 kDa) expression (P<0.001). Incubation of monocytic THP-1 cells with 1 ng rhOPG/105 cells per 1 mL per 24 hours induced a 2-fold increase in MMP-9 expression (P<0.001), a 1.5-fold increase in MMP-2 activity (P=0.005), and a 2-fold stimulation of IL-6 production in these cells (P=0.02). Finally, secretion of OPG from human AAA explant was abrogated by treatment with the angiotensin II blocker irbesartan, with the reduction in secreted levels averaging 63.0±0.9 ng/mg tissue per 48-hour period. Conclusions—These findings support a role for OPG in the growth of human AAA and suggest a potential benefit for angiotensin II blockade in slowing aneurysm expansion.

Reduced expansion rate of abdominal aortic aneurysms in patients with diabetes may be related to aberrant monocyte–matrix interactions

AIMS Diabetes increases the risk of atherothrombosis, but reduces the risk of abdominal aortic aneurysm (AAA). The reason for this difference is unknown. We examined the role of diabetes and glycation on AAA expansion and extracellular matrix-monocyte interactions. METHODS AND RESULTS We followed 198 patients (20 with diabetes) who had 30-45 mm AAAs with yearly aortic ultrasound for 3 years. Diabetes was independently associated with reduced AAA growth (beta = -0.17, P = 0.01; OR for expansion above median 0.18, 95% confidence interval 0.06-0.57). In vitro incubation of resting human monocytes with glycated bovine serum albumin or monomeric type I collagen increased matrix metalloproteinase (MMP) secretion. In contrast, exposure of activated monocytes to glycated type I collagen lattices induced a marked reduction in MMP and interleukin-6 secretion. This de-activating effect was also demonstrated in cross-linked non-glycated collagen lattices, healthy decellularized aortic media, and decellularized aortic media from diabetes patients with atherosclerosis. In contrast, decellularized aortic media from patients with atherosclerosis, but no diabetes, induced increased MMP secretion. CONCLUSION These findings confirm that the progression of AAA is slower in patients with diabetes and suggest a mechanism by which the aortic media may be protected from degradation in these individuals.

Association Between Osteopontin and Human Abdominal Aortic Aneurysm

Objectives—In vitro and animal studies have implicated osteopontin (OPN) in the pathogenesis of aortic aneurysm. The relationship between serum concentration of OPN and variants of the OPN gene with human abdominal aortic aneurysm (AAA) was investigated. Methods and Results—OPN genotypes were examined in 4227 subjects in which aortic diameter and clinical risk factors were measured. Serum OPN was measured by ELISA in two cohorts of 665 subjects. The concentration of serum OPN was independently associated with the presence of AAA. Odds ratios (and 95% confidence intervals) for upper compared with lower OPN tertiles in predicting presence of AAA were 2.23 (1.29 to 3.85, P=0.004) for the population cohort and 4.08 (1.67 to 10.00, P=0.002) for the referral cohort after adjusting for other risk factors. In 198 patients with complete follow-up of aortic diameter at 3 years, initial serum OPN predicted AAA growth after adjustment for other risk factors (standardized coefficient 0.24, P=0.001). The concentration of OPN in the aortic wall was greater in patients with small AAAs (30 to 50 mm) than those with aortic occlusive disease alone. There was no association between five single nucleotide polymorphisms or haplotypes of the OPN gene and aortic diameter or AAA expansion. Conclusions—Serum and tissue concentrations of OPN are associated with human AAA. We found no relationship between variation of the OPN gene and AAA. OPN may be a useful biomarker for AAA presence and growth.

Animal models of abdominal aortic aneurysm and their role in furthering management of human disease

Abdominal aortic aneurysm is a common degenerative disorder associated with sudden death due to aortic rupture. Current therapy is limited to open surgical repair of the aorta or endovascular placement of covered stents to exclude the abdominal aortic aneurysm from the circulation. A number of different animal models have been developed in order to study abdominal aortic aneurysm in an effort to advance current management deficiencies. Large animal models have been mostly used to assist in developing novel methods to surgically treat abdominal aortic aneurysms. Small animal models, particularly those developed in rodents, have been employed to further the understanding of the mechanisms involved in abdominal aortic aneurysm in order to identify potential new medical treatments. It is expected that findings from these animal models will contribute importantly to new treatments for human abdominal aortic aneurysm. This review explores the animal models which are used in abdominal aortic aneurysm research and highlights their advantages and disadvantages.

Peroxisome proliferator-activated receptor ligands reduce aortic dilatation in a mouse model of aortic aneurysm

OBJECTIVE Osteopontin (OPN) is associated with human abdominal aortic aneurysms (AAA) and in vitro studies suggest that this cytokine is downregulated by peroxisome proliferator-activated receptor (PPAR) ligation. We examined the effect of two PPAR ligands within a mouse model of aortic aneurysm. METHODS At 11 weeks of age apolipoprotein E deficient (ApoE(-/-)) mice were given pioglitazone (n=27), fenofibrate (n=27) or vehicle (n=27) in their drinking water. From 13 weeks of age mice received angiotensin II (1 microg/kg/min) infusion via subcutaneous pumps until death or 17 weeks when the aortas were harvested and maximum aortic diameters were recorded. Suprarenal aortic segments were assessed for OPN concentration and macrophage accumulation. Saline infused mice served as negative controls (n=22). RESULTS Angiotensin II induced marked dilatation in the suprarenal aorta (>2-fold increase compared to controls) associated with upregulation of the cytokines OPN and macrophage infiltration. Suprarenal aortic expansion was significantly reduced by administration of pioglitazone (mean diameter 1.61+/-0.11 mm, p=0.011) and fenofibrate (mean diameter 1.51+/-0.13 mm, p=0.001) compared to the vehicle control group (mean diameter 2.10+/-0.14 mm). Immunostaining for macrophages was reduced in mice treated with pioglitazone (median staining area 6.2%, interquartile range 4.1-7.2, p<0.001) and fenofibrate (median staining area 4.0%, interquartile range 2.2-6.1, p<0.001) compared to mice receiving vehicle control (median staining area 13.2%, interquartile range 8.4-20.0). CONCLUSION These findings suggest the potential value of peroxisome proliferator-activated receptor ligation as a therapy for human AAAs.

Osteoprotegerin Is Associated With Aneurysm Diameter and Proteolysis in Abdominal Aortic Aneurysm Disease

Objective—Serum osteoprotegerin (OPG) concentrations have previously been associated with growth of abdominal aortic aneurysms (AAAs). In vitro experiments showed that OPG promotes matrix metalloprotease (MMP) release from monocytes and vascular smooth muscle cells. We hypothesized that OPG expression is increased in human AAAs and is associated with proteolysis. Methods and Results—AAA biopsies were collected from 329 patients. We assessed the concentrations of OPG, cathepsins A, B, and S as well as the activity of MMP-2 and MMP-9. The AAA wall infiltration by macrophages, lymphocytes, and plasma cells was estimated by immunohistochemistry. The concentration of OPG correlated positively with aortic diameter (<55 mm: 16.1 [5.8–28.7], 55–70 mm: 21.9 [10.2–36.0], >70 mm: 24.0 [13.5–52.9] ng OPG/mg total amount of protein, P=0.020), cathepsin A (r=0.221, P=0.005), B (r=0.384, P<0.001), and S (r=0.467, P<0.001), MMP-2 (r=0.180, P<0.001), MMP-9 (r=0.178, P<0.001), and the number of lymphocytes (P<0.001) and plasma cells (P=0.001). OPG immunostaining was predominantly demonstrated in plasma cells. Conclusion—The concentration of aortic wall OPG is positively associated with established markers of AAA severity and pathogenesis. OPG appeared to be associated with lymphocytes and plasma cells. These human data support previous experimental data suggesting a role for OPG in AAA pathogenesis.

MicroRNA profiling in patients with abdominal aortic aneurysms: the significance of miR-155

AAA (abdominal aortic aneurysm) is a potentially life-threatening late-onset degenerative condition. miRNAs (microRNAs), the small non-coding RNA molecules that regulate gene expression, have been shown previously to be associated with a broad range of human pathologies, including cardiovascular diseases. The aim of the present study was to identify AAA-associated miRNAs potentially contributing to AAA pathology. We analysed the expression of 124 miRNAs within AAA biopsies and serum of ten patients undergoing AAA repair, and serum from ten age- and sex-matched subjects without AAA, using the FlexmiR™ MicroRNA Assay. RNA extracted from the site of main AAA dilatation (AAA body) was compared with that extracted from the macroscopically non-dilated neck of the AAA (AAA neck). Similarly, RNA extracted from the serum of AAA patients (AAA serum) was compared with that extracted from age- and sex-matched controls (control serum). qPCR (quantitative real-time PCR), Western blot analysis and histology were performed using an independent set of six paired AAA body and neck biopsies to examine the validity of findings. Seven miRNAs were up-regulated [>2-fold difference, FDR (false discovery rate) <0.5] within AAA biopsies, of which miR-155 was the most differentially expressed (11.32-fold, FDR=0.414). This finding was confirmed by qPCR with the median relative expression of miR-155 being 3.26 and 0.63 within AAA body and AAA neck biopsies respectively (P=0.031). Circulating miR-155 was also increased in AAA patients compared with controls, with a 2.67-fold up-regulation at borderline significance (FDR=0.554). Two immunologically important miR-155 target genes, CTLA4 (cytotoxic T-lymphocyte-associated protein) and SMAD2, were assessed and found to be significantly down-regulated within AAA bodies compared with AAA necks (P=0.032 and P=0.026) as determined by qPCR and Western blotting respectively. Histology demonstrated dense accumulation of T-lymphocytes within the adventitial and outer medial layers of AAA body, but not neck tissue. The results of the present study suggest that miR-155 is overexpressed in AAA with potential implications in the pathogenesis of the condition.

Interaction between Angiotensin II, Osteoprotegerin, and Peroxisome Proliferator-Activated Receptor-γ in Abdominal Aortic Aneurysm

Background and Aims: Osteoprotegerin (OPG) has been associated with abdominal aortic aneurysm (AAA) expansion. Angiotensin II (AngII) receptor blockade has been shown to reduce OPG expression in human AAA tissue. Interaction between vascular AngII and OPG was further examined using cell culture and the AngII-infused ApoE–/– mouse AAA model. The ability of peroxisome proliferator-activated receptor-γ (PPARγ) activation to target OPG as potential therapy for AAA was also investigated. Methods and Results: Human aortic smooth muscle cells (AoSMC) exposed to AngII exhibited dose-dependent increase in the production OPG. A 3-fold increase in suprarenal aortic concentration of OPG was observed in AngII-infused ApoE–/– mice. AngII type 1 receptor expression in human AAA tissue, and AoSMC in vitro, was stimulated up 4-fold in the presence of OPG. This effect in AoSMC was counteracted in the presence of the PPARγ ligand, pioglitazone. Addition of PPARγ ligand to cultured human AAA explant reduced OPG secretion by 60% and tissue concentration of OPG and metalloproteinase 9 by 2- and 3-fold, respectively. Administration of pioglitazone to AngII-infused ApoE–/– mice significantly reduced aortic concentrations of OPG and metalloproteinase 9. Conclusions: These data support an interaction between AngII and OPG in aneurysm formation. Activation of PPARγ may have a role in treatment of AAA.

Role of homocysteine in aortic calcification and osteogenic cell differentiation.

BACKGROUND The role of homocysteine in atherosclerosis is unclear. We examined the relationship between plasma homocysteine and infrarenal aortic calcification, the presence of homocysteine in human atheroma and the influence of homocysteine on osteogenic differentiation in vitro. METHODS AND RESULTS In 194 patients with symptomatic peripheral artery disease or abdominal aortic aneurysm, fasting plasma total homocysteine was independently associated with the severity of infrarenal aortic calcification measured by Computer Tomography Angiography (odds ratio 1.91, 95% confidence interval 1.17-3.21 for calcification >or=median). Homocysteine was identified in all 60 atheroma biopsies from 16 patients undergoing endarterectomy, and concentrations were significantly greater in the calcified biopsies (p=0.003). In vitro studies demonstrated that 100 micromol/L homocysteine doubled the calcium deposition by mesenchymal stem cells during 16 days incubation in osteogenic medium (74+/-4 compared to 42+/-5 microg calcium/well without homocysteine, p<0.001). Homocysteine also stimulated monocytic THP1 cells to promote aortic smooth muscle cell calcification as evidenced by significant higher calcium deposition and alkaline phosphatase activity compared to incubation without homocysteine (p<or=0.05). CONCLUSIONS Homocysteine plays an important role in vascular calcification via multiple mechanisms. The presence of homocysteine in atheroma and its ability to enhance osteogenic cell differentiation may partly explain the association of homocysteine with atherosclerotic events.

Association of PPARγ allelic variation, osteoprotegerin and abdominal aortic aneurysm

Objective  We have previously demonstrated high concentrations of the glycoprotein osteoprotegerin (OPG) in biopsies of abdominal aortic aneurysm (AAA), and demonstrated that ligation of the nuclear receptor peroxisome proliferator‐activated receptor gamma (PPARγ) downregulates OPG in vitro and within a mouse model. The aims of this study were to assess the associations between circulating concentrations of OPG, polymorphisms of the gene encoding PPARγ (PPARG), AAA presence and growth.