Angiopoietin-2 blocking antibodies reduce early atherosclerotic plaque development in mice.

OBJECTIVE: Angiopoietin-2 (Ang-2) blocking agents are currently undergoing clinical trials for use in cancer treatment. Ang-2 has also been associated with rupture-prone atherosclerotic plaques in humans, suggesting a role for Ang-2 in plaque stability. Despite the availability of Ang-2 blocking agents, their clinical use is still lacking. Our aim was to establish if Ang-2 has a role in atheroma development and in the transition of subclinical to clinically relevant atherosclerosis. We investigated the effect of antibody-mediated Ang-2 blockage on atherogenesis after in a mouse model of atherosclerosis. METHODS: Hypercholesterolemic (low-density lipoprotein receptor(-/-) apolipoprotein B(100/100)) mice were subjected to high-cholesterol diet for eight weeks, one group with and one group without Ang-2 blocking antibody treatment during weeks 4-8.To enhance plaque development, a peri-adventitial collar was placed around the carotid arteries at the start of antibody treatment. Aortic root, carotid arteries and brachiocephalic arteries were analyzed to evaluate the effect of Ang-2 blockage on atherosclerotic plaque size and stable plaque characteristics. RESULTS: Anti-Ang-2 treatment reduced the size of fatty streaks in the brachiocephalic artery (-72%, p < 0.05). In addition, antibody-mediated Ang-2 blockage reduced plasma triglycerides (-27%, p < 0.05). In contrast, Ang-2 blockage did not have any effect on the size or composition (collagen content, macrophage percentage, adventitial microvessel density) of pre-existing plaques in the aortic root or collar-induced plaques in the carotid artery. CONCLUSIONS: Ang-2 blockage was beneficial as it decreased fatty streak formation and plasma triglyceride levels, but had no adverse effect on pre-existing atherosclerosis in hypercholesterolemic mice.

Nanoparticle-mediated delivery of pitavastatin inhibits atherosclerotic plaque destabilization/rupture in mice by regulating the recruitment of inflammatory monocytes.

BACKGROUND: Preventing atherosclerotic plaque destabilization and rupture is the most reasonable therapeutic strategy for acute myocardial infarction. Therefore, we tested the hypotheses that (1) inflammatory monocytes play a causative role in plaque destabilization and rupture and (2) the nanoparticle-mediated delivery of pitavastatin into circulating inflammatory monocytes inhibits plaque destabilization and rupture. METHODS AND RESULTS: We used a model of plaque destabilization and rupture in the brachiocephalic arteries of apolipoprotein E-deficient (ApoE(-/-)) mice fed a high-fat diet and infused with angiotensin II. The adoptive transfer of CCR2(+/+)Ly-6C(high) inflammatory macrophages, but not CCR2(-/-) leukocytes, accelerated plaque destabilization associated with increased serum monocyte chemoattractant protein-1 (MCP-1), monocyte-colony stimulating factor, and matrix metalloproteinase-9. We prepared poly(lactic-co-glycolic) acid nanoparticles that were incorporated by Ly-6G(-)CD11b(+) monocytes and delivered into atherosclerotic plaques after intravenous administration. Intravenous treatment with pitavastatin-incorporated nanoparticles, but not with control nanoparticles or pitavastatin alone, inhibited plaque destabilization and rupture associated with decreased monocyte infiltration and gelatinase activity in the plaque. Pitavastatin-incorporated nanoparticles inhibited MCP-1-induced monocyte chemotaxis and the secretion of MCP-1 and matrix metalloproteinase-9 from cultured macrophages. Furthermore, the nanoparticle-mediated anti-MCP-1 gene therapy reduced the incidence of plaque destabilization and rupture. CONCLUSIONS: The recruitment of inflammatory monocytes is critical in the pathogenesis of plaque destabilization and rupture, and nanoparticle-mediated pitavastatin delivery is a promising therapeutic strategy to inhibit plaque destabilization and rupture by regulating MCP-1/CCR2-dependent monocyte recruitment in this model.

Heterogenic endothelial responses to inflammation: role for differential N-glycosylation and vascular bed of origin.

BACKGROUND: Endothelial cell responses during inflammation are heterogeneous and key for selectivity in how leukocytes hone in on specific sites and why vascular diseases are highly bed specific. However, mechanisms for this specificity remain unclear. METHODS AND RESULTS: Here, we exposed human endothelial cells isolated from 5 systemic arterial beds from 1 donor (to overcome donor-to-donor genetic/epigenetic differences), the umbilical vein, and pulmonary microvasculature to TNF-α, LPS, and IL-1ß and assessed acute (ERK1/2 and p65) and chronic (ICAM-1, VCAM-1 total and surface expression) signaling responses and assessed changes in surface N-glycans and monocyte adhesion. Significant diversity in responses was evident by disparate changes in ERK1/2 and p65 NF-κB phosphorylation, which varied up to 5-fold between different cells and in temporal and magnitude differences in ICAM-1 and VCAM-1 expression (maximal VCAM-1 induction typically being observed by 4 hours, whereas ICAM-1 expression was increased further at 24 hours relative to 4 hours). N-glycan profiles both basally and with stimulation were also bed specific, with hypoglycosylated N-glycans correlating with increased THP-1 monocyte adhesion. Differences in surface N-glycan expression tracked with dynamic up- or downregulation of α-mannosidase activity during inflammation. CONCLUSIONS: These results demonstrate a critical role for the vascular bed of origin in controlling endothelial responses and function to inflammatory stimuli and suggest that bed-specific expression of N-linked sugars may provide a signature for select leukocyte recruitment.

Effect of S-aspirin, a novel hydrogen-sulfide-releasing aspirin (ACS14), on atherosclerosis in apoE-deficient mice.

Hydrogen sulfide (H(2)S) is a novel gaseous mediator that plays important roles in atherosclerosis. The present study investigated the effect of a novel H(2)S-releasing aspirin, ACS14 (2-acetyloxybenzoic acid 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl ester), on atherosclerotic plaques in fat-fed apoE(-/-) mice and the underlying mechanism with respect to CX3C chemokine receptor 1 (CX3CR1) in macrophages. Mouse macrophage cell line RAW264.7 or mouse peritoneal macrophages were preincubated with aspirin (50, 100 or 200µM), ACS14 (50, 100 or 200µM) or vehicle for 6h, and then stimulated with interferon (IFN)-γ (500U/ml) or lipopolysaccharide (LPS; 10µg/ml) for 12h. ACS14, but not aspirin, dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 expression and CX3CR1-mediated chemotaxis in macrophages. The inhibitory effect of ACS14 on CX3CR1 expression was abolished by pretreatment with GW9662, a selective peroxisome proliferator-activated receptor (PPAR)-γ antagonist, suggesting that suppression of macrophage CX3CR1 expression by ACS14 is PPAR-γ dependent. Eight-week-old male apoE(-/-) mice received intraperitoneal ACS14 (15 or 30µmol/kg/day) or aspirin (15 or 30µmol/kg/day) 4 weeks after fat feeding. Twelve weeks after ACS14 or aspirin treatment, mice were sacrificed to evaluate the extent of atherosclerosis and CX3CR1 expression in brachiocephalic artery (BCA). We found that ACS14, but not aspirin, significantly downregulated CX3CR1 expression in atherosclerotic plaques. ACS14 considerably impeded the formation and development of atherosclerosis as compared to a molar equivalent dose of aspirin. These data indicate that ACS14 may prevent the progression of atherosclerosis by downregulating macrophage CX3CR1 expression via a PPAR-γ-dependent mechanism.