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The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.

Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.

Anatomic and functional changes after either a permanent left anterior descending coronary artery occlusion (PO) or 2 h of occlusion followed by reperfusion (OR) in C57BL/6 mice were examined and compared with those in sham-operated mice. Both interventions generated infarcts comprising 30% of the left ventricle (LV) measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. Serial follow-up revealed that the ventricular ejection velocity and filling velocity returned to the levels of the sham-operated controls in the OR group at 2 wk and remained there; in contrast, PO animals continued to display suppression of both systolic and diastolic function. In contrast, ejection fractions of PO and OR animals were depressed equivalently (50% from sham-operated controls). Anatomic reconstruction of serial cross sections revealed that the percentage of the LV endocardial area overlying the ventricular scar (expansion ratio) was significantly larger in the PO group vs. the OR group (18 ± 1.7% vs. 12 ± 0.9%, P < 0.05). The septum that was never involved in the infarction had a significantly ( P < 0.002) increased mass in PO animals (22.5 ± 1.08 mg) vs. OR (17.8 ± 1.10 mg) or sham control (14.8 ± 0.99 mg) animals. Regression analysis demonstrated that the extent of septal hypertrophy correlated with LV expansion ratio. Thus late reperfusion appears to reduce the degree of infarct expansion even under circumstances in which it no longer can alter infarct size. We suggest that reperfusion promoted more effective ventricular repair, less infarct expansion, and significant recovery or preservation of ventricular function.

The purpose of this study was to determine the effects of stent placement on the underlying arterial morphology and the relations of stent-vessel wall interactions with subsequent neointimal formation in an atherosclerotic artery. Seven New Zealand White rabbits with experimentally induced atherosclerosis underwent balloon angioplasty ( n = 7) and stent placement after balloon angioplasty ( n = 7) in the iliac arteries. Histologic analysis of the treated arteries was performed at 28 days to assess device interactions with the artery and the pattern of the neointimal response. The area within the external elastic lamina of the stented vessels was 66% greater than the arteries with balloon angioplasty alone ( p = 0.001) which contributed to a significantly greater late lumen area (3.33 ± 0.51 mm 2 versus 1.33 ± 0.20 mm 2 , p = 0.0028). Neointimal thickness was measured at 220 stent wire sites from 21 sections of stented arteries of which 139 (63%) had underlying plaque and 81 (37%) were adjacent to normal media. Rupture of the internal elastic lamina (IEL) occurred at only 9 (11%) of the 81 stent wire sites over normal media. The mean neointimal thickness was 0.16 ± 0.01 mm for all stent wire sites. The neointimal thickness was greater at the stent wire sites with underlying plaque (0.23 ± 0.01 mm) than at the stent wire sites adjacent to normal media (0.08 ± 0.01 mm) or at sites with rupture of the internal elastic lamina (0.16 ± 0.02 mm, p = 0.0001). The degree of neointimal formation within the stents strongly correlated with the area of the underlying atherosclerotic plaque (r = 0.76, p = 0.0007) and the extent of plaque or medial compression by the struts (r = 0.90, p = 0.006). The present study characterizes stent interactions in a model commonly employed to evaluate novel therapies for the prevention of restenosis. The neointimal response was influenced by the local arterial morphology and correlated with the extent of plaque or medial compression by the stent. These data may be useful for future studies in this model and understanding the mechanism of in-stent restenosis.

The purpose of this study was to identify the optimal staining for cell proliferation in paraffin embedded sections of rabbit and pig ileum. Methods comparing zinc formalin or 10 % buffered formalin, 3 PCNA antibodies (19A2, PC10, MAB424), fixation time intervals at 4, 8,24, and 48 hr, and antigen retrieval solution were developed. Staining of nuclei in rabbit ileum was optimal after 4–8 hr in zinc formalin with the PC10, 19A2, and MAB424 antibodies, without antigen retrieval. All 3 antibodies produced optimal staining of nuclei in pig ileum fixed in zinc formalin 4–8 hr without antigen retrieval.After 4 hr fixation in 10% neutral buffered formalin (NBF), nuclei in both pig and rabbit were lightly stained in comparison to those fixed in zinc formalin. Minimal or no staining was seen at 8, 24, and 48 hr fixation in 10% NBF for rabbit ileum. Weak staining was seen at 28 and 48 hr in 10% NBF for pig ileal nuclei.Antigen retrieval methods did enhance nuclear staining with zinc formalin fixation and 10...

Mixed lineage kinase 7 (MLK7) is a recently identified mitogen-activated protein kinase kinase kinase with enriched expression in skeletal muscle and heart. When over-expressed in cardiac myocytes, MLK7 activates both the p38 and c-Jun N-terminal kinase (JNK) stress-activated pathways and induces a cellular phenotype characteristic of cardiac hypertrophy, including a fetal gene expression pattern and increased protein synthesis. We sought to determine the effect of MLK7 on cardiac function in vivo by generating transgenic (Tg) mice with cardiac restricted over-expression of the enzyme. The mice were viable and demonstrated no visible signs of distress at rest. Microscopic examination of the hearts showed myocardial fibrosis and hypertrophy. Hemodynamic analysis of the Tg mice revealed impaired systolic function and significant diastolic dysfunction. Furthermore, significant mortality was observed in MLK7 Tg mice following 24–48 h of isoproterenol administration. Isoproterenol activation of JNK and p38, but not extracellular signal-regulated kinase, was significantly greater in the MLK7 Tg mice compared to littermate controls. These data indicate that MLK7 is an important signal transducer in cardiac compensation. Simultaneous activation of JNK and p38 by MLK7 may contribute to cardiac decompensation during the periods of acute cardiac stress.

Young mice tolerate myocardial loss after coronary artery ligation (CAL) without congestive heart failure (CHF) signs or mortality. We predicted a CHF phenotype after CAL in aged mice. Left coronary artery ligation produced permanent myocardial infarcts (MI). Mortality was higher in male 14-mo-old C57BL/6N mice (Older mice) than in 2-mo-old mice (Young mice) (16 of 25 Older mice died vs. 0 of 10 Young mice, P < 0.02). After 8 wk, rales, weight loss, and lethargy preceded deaths. Captopril (50 mg · kg−1 · day−1) increased Older mouse survival (6 of 22 died, P < 0.02). Captopril improved systolic function (peak aortic blood velocity) from 76 ± 6% of baseline in untreated Older mice to 93 ± 8% ( P < 0.036). At 24 h, MI comprised 28 ± 4% of the left ventricle in Young mice, surprisingly larger than that in Older mice (18 ± 2%, P < 0.011). Endocardial area underlying the infarct scar was significantly larger in Older mice than in Young mice. Captopril did not reduce expansion but markedly reduced septal hypertrophy. Aging reduces compensatory ability in mice despite smaller acute infarcts. Less effective myocardial repair, greater infarct expansion, and septal hypertrophy are seen with aging. Aging is a more relevant murine model of post-MI heart failure in patients.