Joji Shindo

Granulocyte-Macrophage Colony–Stimulating Factor Prevents the Progression of Atherosclerosis via Changes in the Cellular and Extracellular Composition of Atherosclerotic Lesions in Watanabe Heritable Hyperlipidemic Rabbits

BACKGROUND A cytokine network is involved in atherogenesis. This study was conducted to investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development and composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits. METHODS AND RESULTS GM-CSF (10 microg. kg-1. d-1) was administered to 4-month-old WHHL rabbits (n=9) 5 days a week for 7.5 months, whereas an equal dose of human serum albumin was administered to controls (n=9). The cholesterol levels were not changed significantly by the treatment. Age-matched 4-month-old rabbits (n=7) had atheromatous plaques over 30.7+/-5.7% of the inner surface area of the aortic arch. After treatment, the percentages of surface atheromatous plaques to total aortic arch area were 45.0+/-12.6% in the GM-CSF group and 74.3+/-11.0% in controls (P<0.0001). Histological examination demonstrated that GM-CSF reduced the ratio of intima to media (P<0.01) and cross-sectional areas of atherosclerotic lesions (P<0.0001). Quantitative analysis indicated a marked decrease in the areas of smooth muscle cells (P=0.0001), collagen (P=0.0001), and extracellular lipid deposits (P<0.05) of atheromatous plaques in GM-CSF-treated rabbits compared with controls. The terminal deoxynucleotidyltransferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) method and immunohistochemistry were performed to examine the relationship between decreased atherosclerotic lesions and apoptosis. The percentage of TUNEL-positive cells increased in the GM-CSF group (GM-CSF, 24.1+/-4.4% versus control, 11.6+/-3.2%; P<0.0001). GM-CSF enhanced the apoptosis of smooth muscle cells in the shoulder region and the fibrous cap (P<0.0001), suggesting one of the mechanisms for the antiatherogenic effect. CONCLUSIONS GM-CSF altered the composition of atherosclerotic lesions and reduced the atherosclerosis in WHHL rabbits.

Inhibition of Rho/Rho-kinase signaling downregulates plasminogen activator inhibitor-1 synthesis in cultured human monocytes

Increased production of plasminogen activator inhibitor-1 (PAI-1) in plaques plays a role in the pathogenesis of atherosclerosis. This study was conducted to investigate the effect of blockade of Rho/Rho-kinase signaling on the synthesis of PAI-1 in cultured human peripheral blood monocytes. HMG-CoA reductase inhibitors (statins) and inhibitors of Rho and Rho-kinase were added to monocyte cultures. The levels of PAI antigen and mRNA were determined by Western blotting and RT-PCR, respectively, and PAI-1 expression was assessed by immunohistochemistry. We performed pull-down assays to determine the activity of Rho by measuring the GTP-bound form of Rho A. In unstimulated and lipopolysaccharide (LPS)-stimulated cultured monocytes, statins reduced the levels of PAI-1 antigen and mRNA. The suppressive effects of statins on PAI-1 synthesis were reversed by geranylgeranylpyrophosphate (GGPP) and were mimicked by C3 exoenzyme. Immunohistochemistry confirmed the role of lipid modification by GGPP in suppressive effect of statins in PAI-1 synthesis. Pull-down assays demonstrated that statins decreased the levels of the GTP-bound form of Rho A. Our findings suggest that statins decrease the activity of Rho by inhibiting geranylgeranylation. Moreover, Rho-kinase inhibitors, Y-27632 and fasudil, suppressed the synthesis of PAI-1 in this culture system. We show that inhibition of Rho/Rho-kinase signaling downregulates the synthesis of PAI-1 in human monocytes.

Rho/Rho-kinase is involved in the synthesis of tissue factor in human monocytes

Monocytes and macrophages synthesize tissue factor (TF) which plays a role in thrombogenicity in coronary artery disease. This study was conducted to investigate the effect of Rho/Rho-kinase inhibition on the synthesis of TF in cultured human monocytes. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), C3 exoenzyme and Rho-kinase inhibitors were added to isolated peripheral blood monocytes and the synthesis of TF was assessed by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Rho activity was determined by measuring the GTP-bound form of Rho A. Cerivastatin and pravastatin reduced the levels of TF antigen and mRNA. The suppressive effect of statins on TF synthesis was reversed by geranylgeranylpyrophosphate (GGPP) and the restoring effect of GGPP was eliminated by C3 exoenzyme and Y-27632. Pravastatin decreased the activity of Rho A, suggesting that the suppression of TF synthesis by statins is mediated via inhibition of the geranylgeranylation of Rho. Moreover, inhibition of Rho and Rho-kinase downregulated the synthesis of TF. Our results suggest that Rho/Rho-kinase signaling is involved in the synthesis of TF in human monocytes and that inhibition of Rho/Rho-kinase may be useful for treating thrombogenicity in coronary artery disease.

Increased plasminogen activator inhibitor-1 and apolipoprotein (a) in coronary atherectomy specimens in acute coronary syndromes.

BACKGROUND Although increased tissue factor expression is known in vulnerable plaques, there is no reported study to compare plaque fibrinolysis in stable and unstable plaques. This study investigates the extent of plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein (a) [apo(a)] in the plaques of different types of coronary artery disease as well as the correlation between these molecules and infiltration of macrophages to plaques. METHODS Using immunohistochemical staining, we examined PAI-1 expression and apo(a) deposition in coronary atherosclerotic specimens obtained by directional coronary atherectomy from 19 patients with acute myocardial infarction (AMI), 12 with unstable angina pectoris (UAP), and 13 with stable angina pectoris (SAP). The percentages of the total areas of specimens stained with PAI-1 or apo(a) were estimated by an NIH image program. The proportion of macrophages as a percentage of all cells in plaques was calculated as the macrophage density. RESULTS We found significantly higher percentages of total areas of specimens stained with PAI-1 in AMI (25.5 +/- 8.6%, P < 0.001) and UAP (22.2 +/- 10.4%, P < 0.005) than in SAP (9.5 +/- 5.0%), as well as with apo(a) (AMI; 11.7 +/- 7.1%, P < 0.005, UAP; 11.1 +/- 5.5%, P < 0.01 versus SAP; 3.9 +/- 1.5%). Linear regression analysis of all the samples showed a correlation between PAI-1 or apo(a) and macrophage density (PAI-1: r = 0.75, P < 0.001 and apo(a): r = 0.56, P < 0.001). CONCLUSIONS Our results suggest a possible contribution of increased PAI-1 and apo(a) in plaques to the pathogenesis of acute coronary syndromes including impaired fibrinolysis.