Roles of thromboxane A(2) and prostacyclin in the development of atherosclerosis in apoE-deficient mice

Abstract

Production of thromboxane (TX) A2 and PG I2/prostacyclin (PGI2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE-/-TP-/- mice exhibited a significant delay in atherogenesis, and apoE-/-IP-/- mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE-/-IP-/- mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE-/-TP-/- mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE-/-IP-/- and apoE-/-TP-/- mice, respectively, than in those of apoE-/- mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE-/-IP-/- mice than in either apoE-/-TP-/- or apoE-/- mice. We conclude that TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Figure 1

Generation and atherosclerotic lesions of apoE^_/_TP^_/_ and apoE^_/_IP^_/_ mice. (A) Strategy for PCR analysis of WT and targeted alleles of TP and IP. Primers are shown by arrowheads. Amplified fragments are shown by broken lines. Neo, neomycin-resistance gene. (B) Representative PCR for TP and IP alleles of apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice. (C) Representative oil red O staining of aortic sinus sections of apoE^_/_ (middle), apoE^_/_TP^_/_ (lower), and apoE^_/_IP^_/_ (upper) mice. Scale bar: 200 μm. (D) Time course of atherosclerotic lesion development in apoE^_/_ (open circles), apoE^_/_TP^_/_ (filled circles), and apoE^_/_IP^_/_ (filled squares) mice. Data are means ± SEM (n = 10 for 15-week-old apoE^_/_ and apoE^_/_IP^_/_ and 20-week-old apoE^_/_ and apoE^_/_TP^_/_ male mice; n = 6 for 15-week-old apoE^_/_TP^_/_, 20-week-old apoE^_/_IP^_/_, and 30-week-old apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice). *P < 0.05 and **P < 0.01 versus apoE^_/_ mice. (E) Representative Sudan IV staining of en face preparations of aortas from apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice at 20 weeks of age. Scale bars: 2 mm. (F) Quantification of en face atherosclerotic lesions in apoE^_/_, apoE^_/_TP^_/_ and apoE^_/_IP^_/_ mice at 20 weeks of age. Data are means ± SEM (n = 5 each). *P < 0.05 and **P < 0.01 for bracketed comparisons. (G) Representative hematoxylin and eosin staining of innominate artery sections of apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice at 45 weeks of age. Scale bar: 20 μm. (H) Quantitative analysis of innominate atherosclerotic areas in apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice at 45 weeks of age. Data are means ± SEM (n = 10 each). *P < 0.05 and **P < 0.01 for bracketed comparisons.

Figure 2

Effects of TP or IP deficiency on the abundance of macrophages and SMCs and EC integrity in aortic arch lesions of apoE-deficient mice at 20 weeks of age. (A) Representative immunostaining of macrophages and SMCs in aortic arch lesions of apoE^_/_ (left panels), apoE^_/_TP^_/_ (middle panels), and apoE^_/_IP^_/_ (right panels) mice. Lesions were stained with specific antibodies for macrophages (MOMA-2; upper) and for SMCs (α-actin; lower). White arrowheads indicate the external elastic lamina. Scale bars: 20 μm. (B and C) Quantitative analysis of the abundance of macrophages (B) and SMCs (C) in aortic arch lesions of apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice. Data are means ± SEM (n = 10 each). (D) Representative immunostaining of ECs in aortic arch lesions of apoE^_/_ (left panels), apoE^_/_TP^_/_ (middle panels), and apoE^_/_IP^_/_ (right panels) mice. Cross-sections were stained with specific antibodies for ECs (vWF, upper, and PECAM-1, red, lower) and SMCs (α-actin, green, lower). Black and white arrowheads indicate the site of endothelial disruption. Scale bars: 20 μm. (E) Quantitative analysis for endothelial integrity in aortic arch lesions of apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice by measurement of the vWF-positive signals overlying aortic lesions. Data are means ± SEM (n = 10 each). *P < 0.05 for bracketed comparisons. (F) Representative en face staining of aortic arch lesions of apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice. En face staining of aortic arch lesions with silver nitrate or anti_PECAM-1 was performed. Scale bars: 10 μm. (G) Representative scanning electron micrographs of aortic arches of apoE^_/_IP^_/_ mice. The right panel shows a higher magnification of the boxed area in the left panel. Scale bars: 50 μm.

Figure 3

Effects of TP or IP deficiency on ICAM-1 and PECAM-1 expression in the ECs overlying atheromatous lesions of apoE-deficient mice. (A) Representative immunostaining for ICAM-1 and PECAM-1 in cross-sections from apoE^_/_ (left panels), apoE^_/_TP^_/_ (middle panels), and apoE^_/_IP^_/_ (right panels) mice. Mice were sacrificed at 20 weeks of age. Cross-sections were stained with specific antibodies against ICAM-1 (red; lower panels), PECAM-1 (red; upper panels), and smooth muscle α-actin (green). Scale bars: 20 μm. (B) Representative immunostaining of aortic arch lesions and neighboring intact areas for ICAM-1 and PECAM-1 in en face preparations of apoE^_/_ (left panels), apoE^_/_TP^_/_ (middle panels) and apoE^_/_IP^_/_ (right panels) mice. En face preparations were stained with specific antibodies against ICAM-1 (middle panels) and PECAM-1 (upper panels). Mice were sacrificed at 20 weeks of age. Scale bars: 10 μm. Merge, merged images. (C and D) Quantification of ICAM-1 (C) and PECAM-1 (D) expression in ECs overlying aortic arch lesions and neighboring intact areas of apoE^_/_, apoE^_/_TP^_/_, and apoE^_/_IP^_/_ mice. Data are means ± SEM (n = 8 each). *P < 0.05 and **P < 0.01 for bracketed comparisons.

Figure 4

Platelet reactivity for thrombin-induced surface expression of P-selectin. (A) Representative histograms of thrombin-induced P-selectin expression in platelets from apoE^_/_ (upper panels), apoE^_/_TP^_/_ (middle panels), and apoE^_/_IP^_/_ (lower panels) mice. Platelets were either left unstimulated or were stimulated with 0.2, 0.3, or 0.4 U/ml of thrombin. They were then labeled with FITC-conjugated anti_P-selectin and were analyzed by flow cytometry. Filled histograms indicate background signal. (B) Quantification analysis. Concentration-dependent effect of thrombin for P-selectin expression was determined in platelets from apoE^_/_ (open circles), apoE^_/_TP^_/_ (filled circles), and apoE^_/_IP^_/_ (filled squares) mice. Data are means ± SEM (n = 9 each). *P < 0.05 and **P < 0.01 versus apoE^_/_ mice.

Figure 5

Intravital microscopy for leukocyte rolling and adhesion. (A) Fluorescence images of rolling and adherent leukocytes. Black and white arrows indicate rolling and adherent leukocytes, respectively. Vessel lumens are outlined by broken lines. Scale bars: 0.1 mm. (B) Quantitative analysis of rolling leukocytes. Data are means ± SEM (n = 5 each). *P < 0.05 versus apoE^_/_ and apoE^_/_TP^_/_ mice. (C) Quantitative analysis for adherent leukocytes as described in B.