The synergistic inhibition of atherogenesis in apoE-/- mice between pravastatin and the sPLA2 inhibitor varespladib (A-002)

Abstract

Secretory phospholipase A2 (sPLA2) activity promotes foam cell formation, increases proinflammatory bioactive lipid levels, decreases HDL levels, increases atherosclerosis in transgenic mice, and is an independent marker of cardiovascular disease. The effects of the sPLA2 inhibitor A-002 (varespladib) and pravastatin as monotherapies and in combination on atherosclerosis, lipids, and paraoxonase (PON) activity in apoE(-/-) mice were investigated. Male apoE(-/-) mice were placed on a 12-week high-fat diet supplemented with A-002 alone or combined with pravastatin. Atherosclerotic lesions were examined for size and composition using en face analysis, Movat staining, anti-CD68, and anti-alpha actin antibodies. Plasma lipids and PON activity were measured. A-002 decreased atherosclerotic lesion area by approximately 75% while increasing fibrous cap size by over 200%. HDL levels increased 40% and plasma PON activity increased 80%. Pravastatin monotherapy had no effect on lesion size but when combined with A-002, decreased lesion area 50% and total cholesterol levels 18% more than A-002 alone. A-002, a sPLA2 inhibitor, acts synergistically with pravastatin to decrease atherosclerosis, possibly through decreased levels of systemic inflammation or decreased lipid levels. A-002 treatment also resulted in a profound increase in plasma PON activity and significantly larger fibrous caps, suggesting the formation of more stable plaque architecture.

Fig. 1.

Effect of A-002 on atherosclerotic lesions in the proximal, ascending, thoracic, and abdominal aorta. The ascending and thoracic aorta was removed and cleared of all connective tissue before being fixed and pinned out. Lipids were stained with Oil Red O (red). En face staining of the thoracic and abdominal aorta from such animals is shown in A. Lesions in aortic root sections are shown in B. Error bars represent ± SEM. C control; HA: high dose A-002; HA+S: high dose A-002 plus pravastatin; LA: low dose A-002; LA+S: low dose A-002 plus pravastatin; S: Pravastatin.

Fig. 2.

Effect of A-002 on atherosclerotic lesion composition in the aortic root. Representative lesion sections from male apoE^−/− treated with A-002 were stained with Movat Pentachrome showing collagen in yellow, smooth muscle cells (SMCs) in red, and proteoglycans in blue (A) (100×). The thickest portion of the fibrous cap is highlighted in red brackets. SMC α actin content of lesions was stained in red, and the thickest portion of the fibrous cap is highlighted in black brackets (B) (100×). The percentage of lesion area consisting of collagen and proteoglycans (C), or fibrous caps (D) were quantitated. Lesional macrophages, stained red, were detected with an anti-CD68 antibody. Atherosclerotic lesion area positive for macrophages was measured and expressed as a percentage of intimal thickening area in each respective section (E) (50×). Error bars represent ± SEM. C: control; HA: high dose A-002; HA+S: high dose A-002 plus pravastatin; LA: low dose A-002, LA+S: low dose A-002 plus pravastatin, S: Pravastatin.

Fig. 3.

Effect of A-002 on plasma lipoprotein fractionation by size exclusion chromatography. A: Pooled plasma samples from 10 male apoE^−/− of each group were fractionated by fast protein liquid chromatography (FPLC). Individual 0.5 ml fractions were collected and used to determine the relative cholesterol and triglyceride content as described in Methods. B: HDL fraction peaks are highlighted. C: Triglyceride levels from each plasma sample fraction are plotted.