Juliana Yano

Pravastatin Treatment Increases Collagen Content and Decreases Lipid Content, Inflammation, Metalloproteinases, and Cell Death in Human Carotid Plaques Implications for Plaque Stabilization

Background —The clinical benefits of lipid lowering with statins are attributed to changes in plaque composition leading to lesion stability, but supporting clinical data from human studies are lacking. Therefore, we investigated the effect of 3 months of pravastatin treatment on composition of human carotid plaques removed during carotid endarterectomy. Methods and Results —Consecutive patients with symptomatic carotid artery stenosis received 40 mg/d pravastatin (n=11) or no lipid-lowering therapy (n=13; control subjects) for 3 months before scheduled carotid endarterectomy. Carotid plaque composition was assessed with special stains and immunocytochemistry with quantitative image analysis. Plaques from the pravastatin group had less lipid by oil red O staining (8.2±8.4% versus 23.9±21.1% of the plaque area, P <0.05), less oxidized LDL immunoreactivity (13.3±3.6% versus 22.0±6.5%, P <0.001), fewer macrophages (15.0±10.2% versus 25.3±12.5%, P <0.05), fewer T cells (11.2±9.3% versus 24.3±13.4%, P <0.05), less matrix metalloproteinase 2 (MMP-2) immunoreactivity (3.6±3.9% versus 8.4±5.3%, P <0.05), greater tissue inhibitor of metalloproteinase 1 (TIMP-1) immunoreactivity (9.0±6.2% versus 3.1±3.9%, P <0.05), and a higher collagen content by Sirius red staining (12.4±3.1% versus 7.5±3.5%, P <0.005). Cell death by TUNEL staining was reduced in the pravastatin group (17.7±7.8% versus 32.0±12.6%, P <0.05). Conclusions —Pravastatin decreased lipids, lipid oxidation, inflammation, MMP-2, and cell death and increased TIMP-1 and collagen content in human carotid plaques, confirming its plaque-stabilizing effect in humans.

Effect of Immunization With Homologous LDL and Oxidized LDL on Early Atherosclerosis in Hypercholesterolemic Rabbits

Although the existence of an immune response against modified lipoproteins in atherosclerosis has been observed in experimental animals as well as in humans, the precise pathophysiological relevance of these findings remains unclear. In this study we determined the effect of an immunization with homologous LDL and copper-oxidized LDL on the formation of atherosclerotic plaque in hypercholesterolemic rabbits. Immunizations were performed at the start of a cholesterol-rich diet and 3 weeks later. After 16 weeks, antibodies against oxidized LDL had developed in rabbits given hypercholesterolemic diet alone, but the titers were increased by twofold in rabbits immunized with oxidized LDL as well as in rabbits immunized with LDL, suggesting that the LDL had also become oxidized during the preparation and/or immunization procedure. Immunization with LDL and oxidized LDL reduced atherosclerotic lesions in the proximal aorta by 74% (P < .05) and 48% (P = NS), respectively. The cellular composition of the lesions was not affected by the immunizations. These results support the hypothesis that an immune response against modified LDL has a protective effect against the development of early atherosclerotic lesions.

Intermittent brief periods of ischemia have a cumulative effect and may cause myocardial necrosis.

We investigated the effects of brief intermittent periods of ischemia on myocardial viability. Brief periodic coronary occlusions were produced up to 18 times by inflating and deflating the balloon of an intracoronary #2F catheter for periods of 15, 10 or 5 minutes, followed by 15‐minuteperiods of reperfusion.Creatine kinase (CK) release, triphenyl tetrazolium chloride staining, and light and electron microscopy were used to detect the presence of myocardial necrosis. For the study of CK release, blood was taken from the great cardiac vein and the aorta before and at 5-minute intervals during each left anterior descending coronary occlusion, as well as during and 1, 5, 10 and 15 minutes after balloon deflation. In seven of 24 dogs with 15-minute occlusions, in five of 21 dogs with 10-minute occlusions, and in three of 32 dogs with 5-minute occlusions, small but distinct areas of subendocardial necrosis were present. In all dogs with morphologic proof of necrosis, there was periodic release of CK into the great cardiac vein, which peaked immediately after reperfusion, reflecting CK washout. Thus, brief periods of ischenia, which when single do not cause necrosis, have a cumulative effect and may cause myocardial necrosis. This mechanism of necrosis may be relevant clinically in patients with frequent anginal episodes. Since many dogs of this study did not have any myocardial necrosis, the findings also suggest that intermittent repefdusion has a beneficial effect and may prevent necrosis, even when total occlusion time exceeds 200 minutes.

Differential Effects of Green Tea–Derived Catechin on Developing Versus Established Atherosclerosis in Apolipoprotein E–Null Mice

Background—Oxidative stress has been implicated in vascular injury and atherogenesis, and antioxidant treatment has shown favorable results in preclinical studies. Despite this, antioxidant therapy has failed to show benefit in clinical trials. Failure of antioxidants in clinical trials may be partly because such therapy is started after atherosclerosis is already well established, whereas the benefits in animal models may be results from early initiation of antioxidants while atherosclerosis is still evolving. Methods and Results—To test this hypothesis, we evaluated the effect of epigallocatechin gallate (EGCG), the main antioxidant derived from green tea, on evolving and established atherosclerotic lesions in hypercholesterolemic apolipoprotein E–null mice. Established native aortic atherosclerotic lesions and evolving atherosclerotic lesions produced by periadventitial cuff injury to carotid arteries were assessed in mice after 21 and 42 days of treatment with daily intraperitoneal injections of EGCG (10 mg/kg) or PBS. EGCG treatment resulted in an increase in the antioxidant capacity in local vascular tissue and systemic circulation and reduced vascular smooth muscle cell proliferation and redox-sensitive gene activation in vitro. EGCG reduced cuff-induced evolving atherosclerotic plaque size at 21 and 42 days by 55% and 73%, respectively, compared with PBS treatment (P <0.05). Conversely, EGCG had no effect on established lesions in the aortic sinuses or the rest of the aorta. Conclusions—Our data suggest that antioxidant EGCG differentially reduces evolving atherosclerotic lesions without influencing established atherosclerosis in the apolipoprotein E–null mice.

Smoking Increases Tissue Factor Expression in Atherosclerotic Plaques Implications for Plaque Thrombogenicity

BACKGROUND Smoking increases the risk of atherothrombotic events. To determine whether smoking influences plaque thrombogenicity, we examined the effect of cigarette smoking and aspirin use on tissue factor (TF) expression in atherosclerotic plaques. METHODS AND RESULTS A total of 23 apoE-/- mice were exposed to cigarette smoke with (n=9) or without (n=14) aspirin treatment. Eleven mice who were exposed to filtered room air served as controls. Aortic root plaques of mice exposed to smoke had higher immunoreactivity for TF (14+/-4% versus 6.4+/-3%; P=0.0005), vascular cell adhesion molecule-1 (15+/-4% versus 5+/-2%; P=0.002), and macrophages (16+/-5% versus 6+/-2%; P=0.002) compared with nonsmoking controls. Aspirin treatment attenuated smoking-induced changes in plaque composition. In human plaques obtained by carotid endarterectomy, TF immunoreactivity (8+/-5% versus 2+/-2%; P=0.0002) and activity (P=0. 03) were higher in the plaques from smokers (n=28) than those from nonsmokers (n=28). Aspirin use was associated with reduced TF expression in smokers (9+/-8% versus 3+/-4%; P=0.0017). CONCLUSIONS Our results suggest increased plaque TF expression and thrombogenicity as a novel mechanism for the increased risk of atherothrombotic events in smokers. Treatment with aspirin may reduce TF expression.

Differential Effects of Apolipoprotein A-I–Mimetic Peptide on Evolving and Established Atherosclerosis in Apolipoprotein E-Null Mice

Background—Apolipoprotein (apo) A-I and apoA-I–mimetic peptides showed promise to prevent atherosclerosis development. Using a bypassed vein graft model in apoE-null mice, we evaluated the effects of oral or intraperitoneal administration of an apoA-I–mimetic peptide on evolving atherosclerotic lesions in the vein graft and compared such effects on the established atherosclerotic lesions in aortic sinus in the same mice. Methods and Results—We used apoE-null mice in which a segment of inferior vena cava was grafted into the right carotid artery at 16 weeks of age. Native aortic atherosclerotic lesions (established atherosclerosis) and vein-graft atherosclerotic lesions (evolving atherosclerosis) were assessed 4 weeks after daily oral (0.3 mg/mL) or intraperitoneal (50 &mgr;g in 200 &mgr;L saline) administration of an apoA-I–mimetic peptide, D4F. Mice receiving saline or water without D4F served as controls. Both oral and intraperitoneal administration of D4F reduced vein-graft atherosclerotic (evolving lesions) plaque size by 43% and 42%, plaque lipid by 70% and 49%, and macrophage immunoreactivity by 63% and 62%, respectively, compared with controls. In contrast, D4F had no effect on the native aortic sinus atherosclerotic lesions (established lesions). Conclusions—Oral and intraperitoneal administration of the apoA-I–mimetic peptide D4F significantly reduced rapidly evolving atherosclerotic lesions in vein grafts but not established atherosclerotic lesions in aortic sinus. These observations suggest that the type of atherosclerotic lesions and the time of initiation during the course of lesion evolution modulate the beneficial effects of apoA-I–mimetic peptides on atherosclerosis.

Monoclonal Antibody Against Vascular Cell Adhesion Molecule-1 Inhibits Neointimal Formation After Periadventitial Carotid Artery Injury in Genetically Hypercholesterolemic Mice

Vascular cell adhesion molecule (VCAM)-1 is induced in smooth muscle cells after arterial injury, in which it has been implicated in the recruitment of inflammatory cells to the site of injury. To investigate the effect of hypercholesterolemia on VCAM-1 induction after injury and the role of VCAM-1 in neointimal response to injury, we injured the carotid artery of wild-type and apolipoprotein E null (KO) mice fed normal and high cholesterol chow. We demonstrate a graded response of VCAM-1 induction as well as monocyte/macrophage infiltration by immunohistochemistry 3 days after injury that correlated with increasing circulating cholesterol levels. Three weeks after injury, KO mice fed high cholesterol chow (KO HC group) had a significantly greater neointimal formation compared with wild-type and KO mice fed normal chow (P<0.05). Inhibition of VCAM-1 function in the KO HC group by monoclonal antibody treatment significantly reduced monocyte/macrophage infiltration and neointimal formation. There was reduced alpha-actin expression in KO HC mice 7 days after injury that was partially inhibited by VCAM-1 antibody treatment. Cell migration in an in vitro injury model was partially inhibited by monoclonal VCAM-1 antibody treatment. We propose an additional role for VCAM-1 in smooth muscle cell activation and neointimal formation after injury.

Inhibitory Effect on Arterial Injury-Induced Neointimal Formation by Adoptive B-Cell Transfer in Rag-1 Knockout Mice

We investigated the effect of B-cell reconstitution in immune-deficient Rag-1 knockout (KO) mice subjected to arterial injury. After 21 days, injury induced a 4- to 5-fold increase in neointimal formation in Rag-1 KO mice fed normal chow compared with wild-type (WT) mice (0.020±0.0160 [n=8] versus 0.0049±0.0022 [n=8] mm2, respectively;P <0.05) and in western-type diet–fed Rag-1 KO mice compared with WT mice (0.0312±0.0174 [n=7] versus 0.0050±0.0028 [n=6] mm2, respectively;P <0.05). To investigate the role of B cells in response to injury, Rag-1 KO mice were reconstituted with B cells derived from the spleens of WT mice, with donors and recipients on the same diet. Reconstitution of Rag-1 KO mice with B cells from WT mice (both fed normal chow) reduced neointimal formation compared with the effect in unreconstituted Rag-1 KO mice (0.0076±0.0039 [n=9] versus 0.020±0.0160 [n=8] mm2, respectively;P <0.05). Reconstitution of Rag-1 KO mice with B cells from WT mice (both fed a western diet) reduced neointimal formation compared the effect in Rag-1 KO mice (0.0087±0.0037 [n=8] versus 0.0312±0.0174 [n=7] mm2, respectively;P <0.05). Injured carotid arteries from reconstituted Rag-1 KO mice had detectable IgM and IgG, indicating viable transfer of B cells. The results suggest that B cells modulate the response to arterial injury.

CD8+ T Cells Mediate the Athero-Protective Effect of Immunization with an ApoB-100 Peptide

Immunization of hypercholesterolemic mice with selected apoB-100 peptide antigens reduces atherosclerosis but the precise immune mediators of athero-protection remain unclear. In this study we show that immunization of apoE (-/-) mice with p210, a 20 amino acid apoB-100 related peptide, reduced aortic atherosclerosis compared with PBS or adjuvant/carrier controls. Immunization with p210 activated CD8+ T cells, reduced dendritic cells (DC) at the site of immunization and within the plaque with an associated reduction in plaque macrophage immunoreactivity. Adoptive transfer of CD8+ T cells from p210 immunized mice recapitulated the athero-protective effect of p210 immunization in naïve, non-immunized mice. CD8+ T cells from p210 immunized mice developed a preferentially higher cytolytic response against p210-loaded dendritic cells in vitro. Although p210 immunization profoundly modulated DCs and cellular immune responses, it did not alter the efficacy of subsequent T cell dependent or independent immune response to other irrelevant antigens. Our data define, for the first time, a role for CD8+ T cells in mediating the athero-protective effects of apoB-100 related peptide immunization in apoE (-/-) mice.

Smoking increases inflammation and metalloproteinase expression in human Carotid atherosclerotic plaques

Objectives: To evaluate the effects of cigarette smoking on the composition of human carotid endarterectomy plaques. Background: Smoking has been recognized as a major risk factor in atherogenesis. It is believed that smoking contributes to the atherosclerotic process and plaque instability in part by increasing the adherence of macrophages to the vessel wall and inducing the release of proteolytic enzymes. However, data are lacking in humans. Methods: Carotid endarterectomy specimens of 21 smokers and 21 nonsmokers matched for age, gender, and symptoms were immunohistochemically stained with antibodies against CD68 (macrophages [MAC]), macrophage-derived metalloelastase (MMP-12), and tissue inhibitor of metalloproteinase 1 (TIMP-1). Sections were also evaluated for elastin content by van Gieson staining. The stained areas were planimetrically quantified as the percentage of immunopositive tissue area of the total tissue area. Results: Smoking was associated with increased macrophage immunoreactivity (9.1% ± 7.4% vs 3.4% ± 2.9%; P = .003) as well as increased expression of MMP-12 (13.4% ± 6.7% vs 5.5% ± 3.5%; P = .0004). However, plaques from smokers had decreased TIMP-1 expression (7.7% ± 5.7% vs 13.1% ± 8.5%; P = .04) and decreased elastin content (26.9% ± 14.5% vs 38.9% ± 18.4%; P = .02). Conclusions: This study demonstrates that cigarette smoking increases markers of inflammation and tissue destruction in atherosclerotic plaques. This change in plaque composition may at least in part explain the effect of smoking on the instability of human atherosclerotic plaques.