Yoshiki Nishizawa

Serum Osteoprotegerin Levels Are Associated With the Presence and Severity of Coronary Artery Disease

Background—Osteoprotegerin (OPG) is a secretory glycoprotein that belongs to the tumor necrosis factor receptor family. OPG-deficient mice develop severe osteoporosis and medial arterial calcification of the aorta and renal arteries. OPG immunoreactivity was demonstrated in the normal blood vessels and in early atherosclerotic lesions. A recent clinical study suggests that there is a significant correlation between elevated serum OPG levels and cardiovascular mortality. We examined whether serum OPG levels are associated with the progression of coronary artery disease (CAD). Methods and Results—Serum OPG levels were examined in 201 patients who underwent coronary angiography because of stable chest pain. The number of diseased vessels was used to represent the severity of CAD. Serum OPG levels were measured by ELISA and were significantly greater in patients with significant stenosis of the coronary arteries than in those without stenosis. As the severity of CAD increased, there was a significant increase in serum OPG levels. Serum OPG levels were 0.94±0.34, 1.04±0.38, 1.19±0.38, and 1.44±0.54 ng/mL (medians 0.91, 0.99, 1.09, and 1.37) for the subjects with normal coronary arteries or luminal irregularities, 1-vessel disease, 2-vessel disease, and 3-vessel disease, respectively. Multivariate logistic regression analysis revealed that serum OPG levels were significantly associated with the presence of CAD [odds ratio, 5.2; 95% confidence interval, 1.7 to 16.0]. Conclusions—Our data show that serum OPG levels are associated with the presence and severity of CAD, suggesting that OPG may be involved in the progression of CAD.

Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages.

Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-&ggr; (IFN-&ggr;) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1&agr;,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of &bgr;-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-&agr; (TNF-&agr;) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-&agr; and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-&ggr; and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-&agr; and OSM.

Matrix Gla protein is associated with coronary artery calcification as assessed by electron-beam computed tomography

Matrix Gla protein (MGP) is an extracellular matrix protein with wide tissue distribution. It has been demonstrated that the expression of MGP is detected not only in the normal blood vessels but also calcified atherosclerotic plaques, and that MGP deficient mice develop extensive arterial calcification. MGP is thought to be a regulator of vascular calcification. A recent clinical study demonstrates the association between polymorphisms of the MGP gene and increased risk of myocardial infarction. However, there are no reports of the relationship between serum MGP levels and coronary artery calcification (CAC). We evaluated the severity of CAC using electron-beam computed tomography (EBCT), and measured serum MGP levels by enzyme-linked immunosorbent assay in 115 subjects with suspected coronary artery disease. CAC scores were correlated with traditional risk factors, such as age, gender, hyper-tension, diabetes, hyperlipidemia and smoking. The serum MGP levels were lower in patients with CAC than in those without CAC (p<0.001). As the severity of CAC increased, there was a significant decrease in serum MGP levels. Serum MGP levels (U/L) were 116.7 +/- 20.3, 104.9 +/- 19.2, 95.2 +/- 15.2 and 82.2 +/- 19.7, (medians 115.5, 105.0, 94.8, and 81.9) for the subjects with normal (CAC score=0), mild (CAC score=1 to 99), moderate (CAC score=100 to 400), and severe (CAC score >400) coronary calcification, respectively. We found that serum MGP levels are inversely correlated with the severity of CAC. These data suggest a possible role for MGP in the development of vascular calcification.

Platelet P-Selectin Expression Is Associated With Atherosclerotic Wall Thickness in Carotid Artery in Humans

Background—Recent genetic animal models reveal important roles of platelet P-selectin on progression of atherosclerosis. In the present study, we examine the relation between platelet P-selectin expression and atherosclerotic parameters in 517 subjects. Methods and Results—Unrelated subjects (n=517; 235 male and 282 female), including 187 with type 2 diabetes, 184 with hypertension, and 366 with hyperlipidemia, were enrolled in the study. P-selectin expression was determined by whole-blood flow cytometry. Arterial stiffness (stiffness index &bgr;) and arterial wall thickness (intima-media thickness [IMT]) were determined by carotid ultrasound. P-selectin expression was significantly and positively correlated with carotid IMT and stiffness index &bgr;. Multiple regression analyses showed that the association of the percentage of P-selectin–positive platelets with carotid IMT was independent of other clinical factors. Moreover, the percentage of P-selectin–positive platelets was higher in subjects with carotid plaque and was an independent factor associated with occurrence of carotid plaque analyzed by multiple logistic regression analysis. Finally, the percentage of P-selectin–positive platelets was positively associated with age, body mass index, systolic and diastolic blood pressure, and HbA1c and inversely associated with HDL cholesterol. Conclusions—Platelet P-selectin is independently associated with atherosclerotic arterial wall changes in human subjects.

Activation of Apoptosis Signal-Regulating Kinase 1 in Injured Artery and Its Critical Role in Neointimal Hyperplasia

Background—Apoptosis signal-regulating kinase 1 (ASK1), recently identified as one of the mitogen-activated protein kinase kinase kinases, is activated by various extracellular stimuli and involved in a variety of cellular function. Therefore, we first examined the role of ASK1 in vascular remodeling. Methods and Results—We used rat balloon injury model and cultured vascular smooth muscle cells (VSMCs). Arterial ASK1 activity was rapidly and dramatically increased after balloon injury. To specifically inhibit endogenous ASK1 activation, dominant-negative mutant of ASK1 (DN-ASK1) was transfected into rat carotid artery before balloon injury. Gene transfer of DN-ASK1 significantly prevented neointimal formation at 14 days after injury. Bromodeoxyuridine labeling index at 7 days after injury showed that DN-ASK1 remarkably suppressed VSMC proliferation in both the intima and the media. We also examined the role of ASK1 in cultured rat VSMCs. Infection with DN-ASK1 significantly attenuated serum-induced VSMC proliferation and migration. We also compared neointimal formation after cuff placement around the femoral artery between mice deficient in ASK1 (ASK1−/− mice) and wild-type (WT) mice. Neointimal formation at 28 days after cuff injury in ASK1−/− mice was significantly attenuated compared with WT mice. Furthermore, we compared the proliferation and migration of VSMCs isolated from ASK1−/− mice with WT mice. Both proliferation and migration of VSMCs from ASK1−/− mice were significantly attenuated compared with VSMCs from WT mice. Conclusions—ASK1 activation plays the key role in vascular intimal hyperplasia. ASK1 may provide the basis for the development of new therapeutic strategy for vascular diseases.

Arterial wall stiffness is associated with peripheral circulation in patients with type 2 diabetes

The prevalence of peripheral vascular disease (PVD) in diabetic patients is manyfold higher than that of age- and sex-matched nondiabetic subjects. This study was designed to evaluate the relationship between quantitatively determined peripheral circulation in the lower extremities and arterial wall thickness or stiffness in 68 patients with type 2 diabetes. Peripheral circulation during treadmill-exercise was monitored by transcutaneous oxygen tension (TcPO2) and was expressed as percentage of post-exercise TcPO2 adjusted by that of pre-exercise (TcPO2 index). Arterial wall thickness (intima-media thickness; IMT) and stiffness (stiffness beta) were measured by ultrasonography. TcPO2 index was negatively (r=-0.350, P=0.0007) correlated with stiffness beta, not with IMT, of the femoral artery. In patients without insulin therapy (n=52), both fasting plasma insulin concentration (r=-0.323, P=0.0023) and HOMA IR, an insulin resistance index, (r=-0.281, P=0.0084) were negatively correlated with TcPO2 index. Multiple regression analyses showed that association of stiffness beta of the femoral artery or HOMA IR with the TcPO2 index was independent of other factors including age, smoking index, ankle brachial pressure index and IMT of femoral artery. Thus, arterial wall stiffness of femoral artery appears to be a major determinant of peripheral circulation in patients with type 2 diabetes.

Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators

Although lipid‐lowering therapy with 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose‐dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon‐γ, 1α,25‐dihydroxyvitamin D3, tumor necrosis factor‐α, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y‐27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.

Calcium in Internal Medicine

1. Clinical Presentation of Derangements of Mineral Metabolism.- 2. Nutritional Needs of Mineral Metabolism.- 3. Calcium Homeostasis.- A Intestinal Absorption of Calcium.- B Renal Handling of Calcium.- C Hypercalcemia and Primary Hyperparathyroidism.- D Hypocalcemia.- 4. Phosphate Homeostasis.- A Intestinal Absorption of Phosphate.- B Renal Handling of Phosphate.- C Hyper- and Hypophosphatemia.- 5. Magnesium Homeostasis.- A Intestinal Absorption of Magnesium.- B Renal Handling of Magnesium.- C Hyper- and Hypomagnesemia.- 6. The Parathyroid Gland.- 7. Parathyroid Hormone Toxicity in Chronic Renal Failure.- 8. Vitamin D Normal Physiology and Vitamin D Therapeutics in Normal Nutrition and Various Disease States.- 9. Bone.- A Normal Structure and Physiology.- B Primary Osteoporosism.- C Secondary Osteoporosis.- D Rickets and Osteomalacia.- E Bone Disease in Chronic Renal Failure.- 10. Organ Diseases Associated with Deranged Calcium Metabolism.- A Calcium and Ageing.- B Hypertension.- C Lipid Disorder.- D Atherosclerosis.- E Central Nervous System.- F Vascular Calcification.

Platelet in progression of atherosclerosis: a potential target in diabetic patients.

Altered platelet function reported in diabetic patients appears to be involved in the pathogenesis of diabetic vascular complications. However until recently, the role of platelets in progression of spontaneous atherosclerosis remained questionable. The original version of the response to injury hypothesis, where deposited platelets at denuded endothelium play key roles in the spontaneous development of atherosclerosis, has been modified dramatically over the past three decades; atherogenesis is now considered a chronic inflammatory process in which monocytes and T cells play central roles. Recently however, evidence has been accumulated that activated platelet contributes to progression of atherosclerosis in apo E-deficient mice. Activated platelets aggregate with leukocytes, release proinflammatory cytokines, chemokines and growth regulatory molecules, resulting in endothelial activation, leukocyte recruitment and altered smooth muscle cell function. Indeed, it has been shown that activated platelets, and their aggregates with leukocytes are found in the circulation of patients with coronary artery diseases. We have recently shown that circulating P-selectin positive platelets, which are higher in diabetic patients than non-diabetic subjects, are significantly and positively associated with carotid atherosclerosis in the large-scale human studies. This review will focus on altered platelet function in diabetic patients, and its implications in the progression of atherosclerosis.