Multifaceted interaction of bone, muscle, lifestyle interventions and metabolic and cardiovascular disease: role of osteocalcin.

Undercarboxylated osteocalcin (ucOC) may play a role in glucose homeostasis and cardiometabolic health. This review examines the epidemiological and interventional evidence associating osteocalcin (OC) and ucOC with metabolic risk and cardiovascular disease. The complexity in assessing such correlations, due to the observational nature of human studies, is discussed. Several studies have reported that higher levels of ucOC and OC are correlated with lower fat mass and HbA1c. In addition, improved measures of glycaemic control via pharmacological and non-pharmacological (e.g. exercise or diet) interventions are often associated with increased circulating levels of OC and/or ucOC. There is also a relationship between lower circulating OC and ucOC and increased measures of vascular calcification and cardiovascular disease. However, not all studies have reported such relationship, some with contradictory findings. Equivocal findings may arise because of the observational nature of the studies and the inability to directly assess the relationship between OC and ucOC on glycaemic control and cardiovascular health in humans. Studying OC and ucOC in humans is further complicated due to numerous confounding factors such as sex differences, menopausal status, vitamin K status, physical activity level, body mass index, insulin sensitivity (normal/insulin resistance/T2DM), tissue-specific effects and renal function among others. Current observational and indirect interventional evidence appears to support a relationship between ucOC with metabolic and cardiovascular disease. There is also emerging evidence to suggest a direct role of ucOC in human metabolism. Further mechanistic studies are required to (a) clarify causality, (b) explore mechanisms involved and

Vasoactive role for angiotensin II type 2 receptors in human radial artery.

Angotensin II type 2 receptors are believed to counter the effects of the angiotensin type 1 receptors and there is no data relating to the co-localisation of either receptor in human diseased arteries. We sought to determine whether AT2R counter the effects of AT1R and immunolocalise both receptors to cells in human diseased arteries. Human radial arteries (RA, n=11) were placed in organ bath chambers and preincubated with the AT2R antagonist PD123319 for twenty minutes before an angiotensin II dose response curve. Immunohistochemistry was performed to identify receptors and pathology was quantified by image analysis software. We observed both receptors in human arteries. Angiogenic blood vessels within occluded arteries expressed both receptors. PD123319 impaired angiotensin II mediated vasoconstriction by 20 percent (n=5, p less than 0.05), however in other arteries, PD123319 exacerbated angiotensin II-mediated vasoconstriction by 60 percent (n=6, p less than 0.01), respectively. We conclude that inhibition of AT2R can enhance or reduce angiotensin II-mediated vasoconstriction. These data indicate that the role of AT2R in human diseased arteries is divergent although the AT2R-mediated vasorelaxation prevails.

High dietary taurine inhibits myocardial apoptosis during an atherogenic diet: association with increased myocardial HSP70 and HSF-1 but not caspase 3.

BACKGROUND AND AIM: Apoptosis is a major cause of myocyte death, and taurine is anti-apoptotic. Heat shock protein 70 (HSP70) (which is regulated by heat shock factor-HSF-1) is also anti-apoptotic, and caspase 3 stimulates the apoptotic pathway. This study investigated whether taurine affects atherogenic diet-induced myocardial apoptosis, and whether HSP70, HSF-1 and caspase 3 are involved. METHODS: New Zealand white rabbits were divided into 3 groups for 4 weeks according to their diet. Group 1 (control) was fed a normal rabbit diet; Group 2 (MC) received a normal rabbit diet with 1% methionine plus 0.5% cholesterol. Group 3 received MC diet + 2.5% taurine (MCT). RESULTS: The atherogenic diet did not affect myocardial HSP70 or HSF-1 protein, but increased myocardial apoptotic nuclei to 40% (p < 0.01) versus 7% in con and 12% in MCT (p < 0.01). However, in MCT, myocardial HSP70 expression increased by 42.7% versus con and MC (p = 0.016), HSF-1 by 12% versus con and MC (p < 0.05), and total nuclei count increased by 37% versus MC (p < 0.05). Caspase 3 subunits remained unchanged in all groups, and HSP70 was increased approximately twofold in endothelial layer of arterioles (p = 0.01). CONCLUSION: This study shows that taurine could reduce myocardial apoptotic nuclei and thus confer myocardial cytoprotection via stimulating myocardial HSP70 via HSF-1 and caspase 3-independent mechanisms.

ACE2 and AT4R are present in diseased human blood vessels.

A growing body of evidence suggests that the angiotensin II fragments, Ang(1-7) and Ang(3-8), have a vasoactive role, however ACE2, the enzyme that produces Ang(1-7), or AT4R, the receptor that binds Ang (3-8), have yet been simultaneously localised in both normal and diseased human conduit blood vessels. We sought to determine the immunohistochemical distribution of ACE2 and the AT4R in human internal mammary and radial arteries from patients undergoing coronary artery bypass surgery. We found that ACE2 positive cells were abundant in both normal and diseased vessels, being present in neo-intima and in media. ACE2 positive immunoreactivity was not present in the endothelial layer of the conduit vessels, but was clearly evident in small newly formed angiogenic vessels as well as the vaso vasorum. Endothelial AT4R immunoreactivity were rarely observed in either normal and diseased arteries, but AT4R positive cells were observed adjacent to the internal elastic lamine in the internal mammary artery, in the neo-intima of radial arteries, as well as in the media of both internal mammary artery and radial artery. AT4R was abundant in vaso vasorum and within small angiogenic vessels. Both AT4R and ACE2 co-localised with smooth muscle cell alpha actin. This study identifies smooth muscle cell alpha actin positive ACE2 and AT4R in human blood vessels as well as in angiogenic vessels, indicating a possible role for these enzymes in pathological disease.

Calcitonin receptor immunoreactivity associated with specific cell types in diseased radial and internal mammary arteries.

AIMS: To determine and quantify calcitonin receptor (CTR) immunoreactivity associated with specific cell types within, and associated with, the endothelial layers, neo-intima, media and vasa vasorum of diseased radial and internal mammary arteries. METHODS AND RESULTS: Immunohistochemistry and anti-CTR antibodies were used to identify positive cells within remnants of diseased human radial (n = 3) and internal mammary arteries (n = 4) that remained after bypass surgery. Three cell types expressed CTR, including endothelial cells, fibroblast-like cells within the neo-intima, and cellular structures aligned with the smooth muscle cells of the media. Other smaller cells within the surrounding parenchyma of the vasa vasorum of diseased vessels and blood-borne cells were also immunoreactive. Immunoquantification of CTR expression (Intensity x Proportional Area) in the endothelium (P < 0.05), neo-intima (P < 0.02) and media (P < 0.03) established a significant statistical correlation (Students' two-tailed t-test) with the ratio of intimal/media thickness. CONCLUSIONS: Increased immunoreactivity developed using anti-CTR antibodies was associated with specific cell types in the endothelial layers, neo-intima, media and vasa vasorum of diseased regions of radial and internal mammary arteries, in which there was an increased intimal/media ratio. Furthermore, CTR+, blood-borne cells present in the vessels of diseased regions suggest recruitment into these surrounding tissues.

Pharmacological characterization of a novel factor Xa inhibitor, FXV673.

FXV673 is a novel, potent, and selective factor Xa (FXa) inhibitor. FXV673 inhibited human, dog, and rabbit FXa with a K(i) of 0.52, 1.41, and 0.27 nM, respectively. FXV673 also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C (aPC), plasmin, and tissue-plasminogen activator (t-PA). FXV673 prolonged plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) in a dose-dependent fashion. In the APTT assays, the concentrations (microM) required for doubling coagulation time were 0.41 (human), 0.65 (monkey), 1.12 (dog), 0.25 (rabbit), and 0.80 (rat). The concentrations (microM) required in the PT assays were 1.1 (human), 1.32 (monkey), 2.31 (dog), 0.92 (rabbit), and 1.69 (rat). A coupled-enzyme assay was performed to measure thrombin activity following prothrombinase conversion of prothrombin to thrombin. FXV673 showed IC(50)s of 1.38 and 2.55 nM, respectively, when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or fresh platelets were used as the phospholipid source for prothrombinase complex formation. It was demonstrated that FXV673 could inhibit further thrombin generation in the prothrombinase complex using PS/PC liposomes. FXV673 dose-dependently prolonged the time to vessel occlusion and inhibited thrombus formation in well-characterized canine models of thrombosis. Interspecies extrapolation (approximately 2.5-fold higher sensitivity for FXa inhibition in human than in dog) suggested that 100 ng/ml of FXV673 would be an effective plasma concentration for clinical studies. Currently FXV673 is undergoing clinical studies to be developed as an antithrombotic agent.

Increased apoptosis in the heart of genetic hypertension, associated with increased fibroblasts.

OBJECTIVE: The present studies were undertaken to identify apoptosis in cardiomyocytes of genetic hypertension and to study the relationship among apoptosis, aging and blood pressure, and the effect of angiotensin-converting enzyme (ACE) inhibitors on apoptosis. METHODS: Apoptosis in the hearts of spontaneously hypertensive rats (SHR) was identified by electron microscopy (EM) and DNA laddering, and quantified from age 3 weeks to 64 weeks in comparison with normotensive rats (WKY). Fibroblasts and protein products of Bcl-2 and Bax were measured by quantitative immunohistochemistry. SHR were treated with ramipril, an ACE inhibitor. RESULTS: The results showed that: (1) ultrastructural characteristics of apoptosis were observed in cardiomyocytes of SHR, with shrinkage of the cell and condensation of the cytoplasm and chromatin. A DNA ladder was shown; (2) a significant increase in apoptosis in SHR began as early as age 4 weeks and reached a plateau at 16 weeks and maintained at high levels up to 64 weeks. Blood pressure (BP) in SHR started to increase significantly at age 5 weeks; (3) fibroblasts were significantly increased in the heart of SHR; (4) the ratio of Bcl-2/Bax was significantly reduced in SHR; and (6) ramipril effectively reduced apoptosis and fibroblasts, and increased the ratio of Bcl-2/Bax. CONCLUSION: Apoptosis occurs in the cardiomyocytes of genetic hypertension although fibroblasts are increased, and a significant, age-dependent increase in apoptosis is observed. The increase in apoptosis occurs before the difference in blood pressure is detectable. The ACE inhibitor ramipril may be useful for prevention of apoptosis in the heart.

The relationship between apoptosis and spontaneous hypertension.

OBJECTIVE: To explore the relationship between apoptosis and spontaneous hypertension, and the effect of angiotensin converting enzyme (ACE) inhibitor on apoptosis. METHODS: Male spontaneously hypertensive rats (SHR) and normotensive control rats (WKY) at different ages were used, meanwhile, the treatment of SHR with ramipril, an inhibitor of ACE was administered orally (1 mg.kg-1.d-1) to SHR from 3 to 10 or from 5 to 10 weeks of age. Apoptosis in cardiomyocytes of SHR was quantified by a maximal labeling (Lmax) method and the characteristic features of apoptosis were identified by electron microscopy (EM), in situ labeling of DNA strand breaks with terminal deoxynucleotidyl transferase mediated dUTP end labeling (TUNEL) and autoradiographic analysis of DNA fragments. RESULTS: The results of a quantitative method showed an age-dependent increase in apoptosis in the cardiac tissues of SHR. A significant increase in DNA breaks occurred as early as 4 weeks and continued to increase up to a plateau at 16 weeks in the cardiac tissue of SHR whereas there was no significant change in apoptosis in WKY up to 64 weeks. Moreover, after the treatment of SHR with ramipril, an inhibitor of angiotensin converting enzyme (ACE), from 3 to 10 or from 5 to 10 weeks of age, the DNA fragmentation as well as blood pressure (BP) was reduced significantly compared with that of untreated SHR (P < 0.01), and similar to that of the control WKY. CONCLUSION: There is a significant increase in the apoptosis of SHR cardiac tissues with increasing age, and ramipril can significantly prevent the increase of apoptosis and in blood pressure, which demonstrates that apoptosis may be involved in the pathogenesis of genetic hypertension. The inhibition of apoptosis as well as hypertension by ACE inhibitors may open a new avenue for developing therapeutic approach for hypertension.

Augmented effects of methionine and cholesterol in decreasing the elastic lamina while thickening the aortic wall in the rat aorta.

1. Patients with an elevated plasma level of either homocysteine or cholesterol are at increased risk of cardiovascular disease. Both methionine, the precursor of homocysteine, and cholesterol are found primarily in the same foods; therefore we investigated the effect of methionine feeding alone, cholesterol feeding alone, and both, on the thickness of the aortic wall and the aortic elastic lamina of normotensive animals.2. Twenty normotensive rats were divided into four groups of five animals. The following diet was administered for 15 weeks: normal chow; normal chow supplemented with 2% methionine; normal chow supplemented with 2% cholesterol; normal chow supplemented with 2% methionine+2% cholesterol.3. The results showed a 3-fold decrease (P<0.003) in the aortic elastic lamina in the 2% methionine group and a 2.5-fold decrease in the 2% cholesterol group compared with the normal chow group. There was a 9-fold (P<0.0003) decrease in the 2% methionine+2% cholesterol group compared with the normal chow group. Furthermore, feeding with methionine plus cholesterol significantly increased aortic wall thickness compared with the methionine group, cholesterol group or control.4. These results demonstrate an augmented effect of cholesterol plus methionine in the deterioration of the aortic elastic lamina, and furthermore, the combination of these two agents increases the thickness of the aortic wall. The results indicate a more important role for these two agents in combination than for either agent alone.

Effect of homocysteine and cholesterol in raising plasma homocysteine, cholesterol and triglyceride levels.

A high plasma homocysteine level is a newly regarded risk factor for coronary artery disease. We report a synergistic effect of homocysteine plus cholesterol feeding on further raising total plasma homocysteine, cholesterol and triglycerides levels than each agent alone, which further enhances the risk of coronary artery disease.

A novel immunohistochemical semiquantitative technique for endothelial constitutive nitric oxide synthase immunoreactivity in rat coronary artery.

It has been difficult to quantify protein production in small pathological specimens by conventional techniques. We describe a new method for semiquantification of immunohistochemical staining, which involves application of the enzyme-labeled avidin (LAB) technique, coupled with an ultra-sensitive and fast chemiluminescent substrate for alkaline phosphatase. The entire procedure can be completed in less than 3 hr. The final step involves X-ray film exposure for 30 min, and the optical density of the subsequent images is examined with a microcomputer imaging device. The optical densities are translated into relative protein concentrations by a reference standard curve, obtained via an immunoblot. To establish a model for semiquantification of endothelial constitutive nitric oxide synthase (eNOS) protein, we compared the coronary arteries of WKY rats fed a normal chow diet to the coronary arteries of WKY rats fed a cholesterol diet. Using this technique, we have found a relative 130-fold decrease in eNOS in the cholesterol-fed group compared to the normal chow-fed group.