A 2- to 6-fold increased risk of coronary heart disease (CHD) is associated with the presence of three or more of the risk factors high triglycerides, low HDL cholesterol, abdominal obesity, hypertension and high fasting glucose, which also identify the metabolic syndrome. Insulin resistance, a key future of the syndrome and related to obesity, particularly abdominal obesity, can lead to glucose intolerance and diabetes. The insulin resistant state can lead to hypertension, through the increased sympathetic activation and the increased re-absorption of sodium and water at the renal tubular level that promotes endothelial dysfunction, and to increased release of free fatty acids from fat cells with reductions in plasma HDL and increases in plasma triglycerides and small dense LDL.

The World Heath Organization defines overweight as a body mass index (BMI) greater than 25, obesity greater than 30, and morbid obesity greater than 40. In the US, 50% of women and 60% of women were obese in year 2000. About 20% of both sexes are obese. Since 1991, overweight has increased by 25% and obesity by 61% in the US. In Japan, in the early 1990s, 20% of people were overweight and 2-3% were obese. Diabetes prevalence in the US increased from 4.9% in 1990 to 7.3% in 2000. Undiagnosed diabetes increases the prevalence to 10%. Diabetes prevalence is similar in Japan.

Fibrates exert a number of beneficial effects on lipid metabolism, hemostasis and atherogenesis, and reduce clinical events in people with atherogenic dyslipidemia and the metabolic syndrome, as shown by clinical trials. Many of the effects of fibrates are mediated by their activity as a PPAR (peroximase proliferatated activated receptor), whose characteristics are outlined in Figure 1. Some effects of PPAR alpha activation on atherosclerosis, an area of active research, are shown in Figure 2. Activation of PPAR alpha induces lipoprotein lipase (LPL) expression leading to increased lipolysis of triglyceride-rich lipoproteins and a decrease in triglycerides and increase in HDL. APO C III expression is inhibited, leading to enhanced LPL-mediated lipolysis, as well as APO I and APO II expression, causing an increase in plasma HDL.

The fibrate gemfibrozil was associated with a 6% increase in HDL, a 31% decrease in triglycerides and no change in LDL cholesterol, compared to placebo, in the VA HDL multicenter trial, stated Rubins of the Minneapolis VA Medical Center. A 22% relative risk reduction (RRR) for CHD death and nonfatal myocardial infarction (MI) was observed (p<0.006). The trial randomized 2,531 men less than 74 years of age with CHD and LDL less than 140 and HDL less than 40 to gemfibrozil or placebo and followed them 5 to 7 years. A 28% RRR with gemfibrozil was seen in the nearly one-half of patients with metabolic syndrome (p=0.001). A more limited drug efficacy was seen in the patients without the metabolic syndrome. A 37% RRR was seen with gemfibrozil in patients with metabolic syndrome and no diabetes. A 35% RRR with gemfibrozil was seen in non-diabetic hyperinsulinemic patients (p=0.036). Interestingly, this is the first report of a therapeutic intervention that prevented clinical cardiovascular events in non-diabetics with hyperinsulinemia.

Glitazones, insulin-sensitizing agents, improve insulin stimulated glucose disposal, may promote insulin inhibition of hepatic glucose production, and have effects on lipid metabolism, hemostasis and atherogenesis. There is no clinical trial evidence that these PPAR gamma activators reduce cardiovascular events in the setting of insulin resistance of metabolic syndrome. Favorable effects of glitazones include inhibition of smooth muscle cell migration and proliferation, inhibition of secretion of inflammatory cytokines by activated monocytes, inhibition of endothelial expression of adhesion molecules, promotion of cholesterol efflux from foam cells and apoptosis of activated macrophages. Potential unfavorable effects are increased in macrophage uptake of oxidized LDL and apoptosis of vascular endothelial cells. Trial data is needed to determine whether the favorable or unfavorable effects are dominant and the efficacy of these agents on reducing cardiovascular events.

Recent work by Shimokawa and colleagues has identified that endothelium-derived hydrogen peroxide is an endothelium-derived hyperpolarizing factor (EDHF). This identification is thought to contribute to the evolving concept of endothelium-derived relaxing factors (EDRF), because, in addition to nitric oxide, another reactive oxygen species served to maintain vascular function and homeostasis in vitro and in vivo.

Endothelial cells play a key role in vascular homeostasis by synthesizing and releasing vasodilating factors, including prostacyclin, nitric oxide (NO) and EDHF. Other important vasodilating factors include adrenomedullin and C-type natriuretic peptide (CNP). The relative contribution of prostacyclin, NO and EDHF to endothelium-dependent relaxation (EDR) is markedly varied, depending on the size of the blood vessels, and can be evaluated experimentally by the inhibitory effect of indomethacin, L-NNA and KCl. This group showed that the contribution of EDHF increased as the vessel size decreased. In contrast, NO seemed to play a major role in large arteries. The same effects were shown in isolated human mesenteric arteries, where EDHF was important in causing bradykinin-induced EDR in microvessels. That NO plays a major role in relatively large arteries, whereas EDHF plays a major role in small resistance arteries is thought to be true in various species and vascular beds.

NO and EDHF synergistically produced EDR in a relatively redundant manner in normal blood vessels in a series of experimental and clinical studies by these investigators. With increasing endothelial injury caused by underlying risk factors, EDR was progressively reduced. Both NO-mediated and EDHF-mediated components were decreased with increased endothelial injury, and were restored with improvement in the endothelial injury. The calcium/calmodulin pathway is required for the synthesis of both NO and EDHF.

Based on these lines of evidence, they hypothesized that EDHF might be a non-NO factor derived from endothelial NO synthase, which they investigated using eNOS-knockout mice. In wild-type mice, EDR to acetylcholine (ACh) was largely mediated by EDHF with some contribution from NO. In contrast, in eNOS knockout mice, the EDR to ACh was significantly reduced, but with some compensatory augmentation by prostaglandin. Importantly, in the knockout mice, the EDHF-mediated EDR was significantly reduced. Endothelium dependent hyperpolarization in response to ACh was significantly reduced in eNOS-knockout mice compared to wild type mice. Vascular smooth muscle relaxation responses to levkromaklium, a direct opener of K channels, were normal in eNOS-knockout mice, and were comparable in both strains. The response to nitroprusside, a NO donor, was rather enhanced in eNOS-knockout mice. These results appear to confirm their hypothesis.

The non-NO factor derived from eNOS was then considered. They examined the inhibitory effect of catalase, which specifically converts hydrogen peroxide (H₂O₂) into water and oxygen, to test their hypothesis that superoxide is dismutated by SOD into H₂O₂, which then may play a role as an endogenous EDHF. These experiments demonstrated that 1) catalase markedly inhibited EDHF-mediated responses, 2) exogenous H₂O₂ elicited similar relaxations and hyperpolarizations, 3) endothelial production of H₂O₂ was noted in wild-type mice but not in eNOS-knockout mice in which EDHF-mediated responses were markedly reduced. Other work has confirmed that H₂O₂ is an endogenous EDHF in porcine coronary microvessels and in human mesenteric arteries, based on the finding that catalase markedly inhibited the bradykinin-induced EDHF-mediated relaxation and hyperpolarization. Also confirmed was that exogenous H₂O₂ mimicked those EDHF-type responses. The results of this work are summarized in Figure 3. The importance of gap junction in the mechanism of EDHF-mediated relaxation and hyperpolarizations has not been confirmed.

In other work, coronary microvessels dilated in a stepwise manner as perfusion pressure was decreased from 70 mm Hg to 30 mmHg, representing autoregulatory coronary vasodilation. L-NNA significantly inhibited this autoregulatory relaxation, which was interestingly further inhibited by catalase. This indicates that both NO and H₂O₂ are involved in coronary autoregulatory vasodilation in vivo.

Endothelial nitric oxide synthase (eNOS) produces nitric oxide when L-arginine or its cofactor BH4 is sufficient for the catalytic activity of eNOS. This eNOS-derived NO acts to protect or maintain vascular structural integrity. However, vascular tissue levels of BH4 are relatively low under pathological conditions, and the mechanisms for this are unclear. Activation of eNOS leads to an "guncoupling of eNOS". The uncoupled eNOS generates superoxide, which impairs vascular function and promotes atherosclerosis. Hence, eNOS has two different effects on vascular structural integrity. In some cases these findings may suggest caution about the recent therapeutic strategy of increasing eNOS expression to treat cardiovascular disease. Seinosuke Kawashima, M.D., of Kobe University, reviewed the research conducted by he and his colleagues that support this working hypothesis.

The role of the eNOS/NO system in vivo using transgenic mice overexpressing eNOS mainly at the level of the endothelium (eNOS-Tg) was studied. In the eNOS-Tg mice, NO production and cGMP concentration were increased.

The effects of eNOS-derived NO inhibit vascular remodeling. In one model, intimal and medial thickening occurred and lumen diameter decreased when the common carotid artery was ligated just below the bifurcation in wild type mice. In contrast, in the eNOS-Tg, intimal and medial thickening was markedly attenuated. Vascular remodeling was increased in both the wild type and transgenic mice with L-NAME. Medial thickening of small pulmonary arterioles produced by three weeks of 10% hypoxia seen in wild type mice was attenuated in eNOS-Tg mice. The ratio of muscular to non-muscularized small pulmonary arterioles was increased with chronic hypoxia in the wild type mice, but was attenuated in the eNOS-Tg mice.

Overexpression of eNOS protects muscle tissue against ischemia and reperfusion. This positive effect in small vessels is at least in part mediated by the maintenance of vascular permeability by eNOS-derived NO. Superoxide production, detected by dehydroethydium staining, was increased in response to 60 minutes of femoral artery occlusion in the wild type animals, but attenuated in the eNOS-Tg. Ischemia and reperfusion increased vascular permeability in wild type mice but was attenuated in ENOS-Tg mice, and tissue damage was attenuated in ENOS-Tg mice.

eNOS-derived NO inhibits inflammation, as shown by an experiment in which marked lung inflammation in response to an injection of LPS was seen in wild type mice but was significantly less in eNOS-Tg mice. Ischemia-induced angiogenesis is promoted by eNOS-derived NO. At 2 weeks, more collateral vessels were seen in ischemic tissue of eNOS-Tg mice compared to wild type mice in response to ligation of the proximal portion of the femoral artery and distal portion of the saphenous artery in the hindlimb.

In contrast to these positive effects, Xi and colleagues showed that eNOS produces superoxide in the absence of L-arginine or the cofactor BH4. BH4 improves depressed endothelium-dependent vasorelaxation (EDR) in diabetes, hypertension, hyperlipidemia and atherosclerosis in animal models and humans. BH4 improved depressed EDR in isolated human coronary arteries from patients with severe atherosclerosis. Superoxide production by the endothelium of the aorta is increased in apoE knockout mice, as shown by Harrison and colleagues. Sepiapterin, a precursor of BH4, abates this superoxide production.

A deficiency of eNOS promotes atherogenesis, as shown by work in eNOS-knockout mice. In apoE knockout mice crossed with eNOS-Tg mice and fed a high-cholesterol Western diet, atherosclerotic lesion formation was augmented. The marked increases in plasma lipid levels were not modified by eNOS overexpression, while blood pressure was lower in study mice, which may be involved with the reduced lesion formation.

Further work confirmed the endothelium to be the main production source for superoxide in the mice aorta, as a result of eNOS overexpression. In aortas from apoE knockout mice overexpressing eNOS, BH4 levels were decreased and BH2, its oxidized form, increased compared to control apoE knockout mice. In the presence of hyperlipidemia, it is likely that BH4 levels are insufficient for the overexpressed eNOS to produce NO. Chronic BH4 treatment increased the BH4 content and decreased the BH2 content in the aorta. Further, superoxide production was decreased in the aorta of apoE knockout mice overexpressing eNOS. In contrast, NO production from aortas was increased with BH4 treatment. The superoxide generated by eNOS in the presence of reduced BH4 availability may promote atherosclerosis lesion formation.

Work by Maemura and colleagues as the University of Tokyo suggests that circadian output genes are in part regulated by the central biological clock with humoral factors or autonomous nervous system. But, these genes may be directly regulated, at least in part, by peripheral clocks in the cardiovascular (CV) system, for example in vascular endothelial cells. Prevention and treatment of CV diseases may benefit from further understanding of this mechanism.

The biologic clock comprises transcriptional-translational feedback loops. The CLOCK-BMAL1 heterodimer binds to the E-box site in the promoter region of Per and Cry genes and transactivates the Per and Cry genes. The Per and Cry genes accumulate in the cytoplasm, and translocate into the nucleus and turn off the transcription of Per and Cry genes by CLOCK-BMAL1 heterodimers. This negative feedback loop occurs over 24 hours. CLOCK, BMAL1 and Per belong to the basic helix-loop-helix (bHLH)/PAS domain transcription factor family, whose features are shown in Figure 4.

CLIF, cycle like factor, was identified by this group in their work to identify transcription factors important for vascular endothelial function, using EPAS1 as bait when screening the HUVEC cDNA library using the yeast two-hybrid system. CLIF is a novel bHLH/PAS protein that shares a high homology with Drosophila CYCLE and its mammalian homologue BMAL1. Another research group cloned the same gene and named it BMAL2. Maemura and colleagues showed that CLIF interacts with EPAS1 and CLOCK, similar to BMAL1, and that a heterodimer of EPAS1 and CLIF are important for hypoxic responses.

Work by this group shows that CLIF and CLOCK plays a role in the biological clock. CLIF is expressed in endothelial cells and neurons in the brain, including the suprachiasmatic nucleus (SCN), the center of the circadian clock, where it functions as a component. It has been demonstrated that clock-related genes are expressed in the SCN and in peripheral organs, suggesting the existence of peripheral oscillators in each organ. Unknown humoral factors synchronize the peripheral and central clocks. Direct regulation of physiologically important circadian output genes may be a function of the peripheral clocks.

A peripheral clock in the CV system was demonstrated by this group in mice on a 12-hour light and dark cycle, in which BMAL1, Per2 and Cry1 exhibited circadian variation in the heart, aorta and kidneys. They hypothesized that the peripheral oscillator may exist in vascular endothelial cells, since certain endothelial functions in regulating vascular tone and fibrinolytic activity show circadian variation. Northern blot analysis clearly demonstrated the circadian oscillation of clock-related gene expression in endothelial cells.

Then they hypothesized that CLOCK and CLIF may regulate the circadian variation of PAI-1 gene expression in endothelial cells. In mouse heart and kidney, circadian variation of PAI-1 mRNA levels with peak evening expression was demonstrated. This pattern is the opposite that seen in humans, perhaps because rodents are nocturnal and humans diurnal. Adenovirus-mediated gene transfer showed that CLOCK overexpression results in a dose-dependent increase in PAI-1 mRNA levels relative to the control cells infected with a GFP-expressing adenovirus. A further increase in PAI-1 mRNA levels occurred with co-infection of an adenovirus expressing CLIF with CLOCK. A 5-fold increase in PAI-1 promoter activity occurred with cotransfection of CLOCK and CLIF using human PAI-1 promoter/luciferase reporter plasmids.

Deletion analysis localized the responsive element between -800 and -549. The two E-box sites, the consensus binding sites of CLOCK and BMAL1, were shown to be important for the CLOCK:CLIF transactivation of the PAI-1 promoter, since mutation in either box decreased transactivation and mutation in both abolished it. The CLOCK:CLIF heterodimer was shown to transactivate the PAI-1 promoter by directly binding to the E-boxes. Per2 and Cry1, the negative components of the biological clock, were shown to suppress the CLOCK:CLIF-mediated transcription of PAI-1, resulting in the circadian oscillation of PAI-1 gene expression.

Their results suggest the presence of a peripheral oscillator in the vascular endothelial cells, comprised of CLOCK, CLIF, Per and Cry, which may regulate the circadian expression of PAI-1 genes as a circadian output gene (Figure 5). In cultured primary rat cardiomyocytes, the rhythmic expression of BMAL1, Per2 and Cry 1 was seen. Figure 6 summarizes their research findings.