Circadian rhythms of risk factors and management in atherosclerotic and hypertensive vascular disease: Modern chronobiological perspectives of an ancient disease.

Atherosclerosis, a chronic inflammatory disease of the arteries that appears to have been as prevalent in ancient as in modern civilizations, is predisposing to life-threatening and life-ending cardiac and vascular complications, such as myocardial and cerebral infarctions. The pathogenesis of atherosclerosis involves intima plaque buildup caused by vascular endothelial dysfunction, cholesterol deposition, smooth muscle proliferation, inflammatory cell infiltration and connective tissue accumulation. Hypertension is an independent and controllable risk factor for atherosclerotic cardiovascular disease (CVD). Conversely, atherosclerosis hardens the arterial wall and raises arterial blood pressure. Many CVD patients experience both atherosclerosis and hypertension and are prescribed medications to concurrently mitigate the two disease conditions. A substantial number of publications document that many pathophysiological changes caused by atherosclerosis and hypertension occur in a manner dependent upon circadian clocks or clock gene products. This article reviews progress in the research of circadian regulation of vascular cell function, inflammation, hemostasis and atherothrombosis. In particular, it delineates the relationship of circadian organization with signal transduction and activation of the renin-angiotensin-aldosterone system as well as disturbance of the sleep/wake circadian rhythm, as exemplified by shift work, metabolic syndromes and obstructive sleep apnea (OSA), as promoters and mechanisms of atherogenesis and risk for non-fatal and fatal CVD outcomes. This article additionally updates advances in the clinical management of key biological processes of atherosclerosis to optimally achieve suppression of atherogenesis through chronotherapeutic control of atherogenic/hypertensive pathological sequelae.

Transcriptional regulation of vascular smooth muscle cell proliferation, differentiation and senescence: Novel targets for therapy.

Vascular smooth muscle cells (SMC) possess a unique cytoplasticity, regulated by transcriptional, translational and phenotypic transformation in response to a diverse range of extrinsic and intrinsic pathogenic factors. The mature, differentiated SMC phenotype is physiologically typified transcriptionally by expression of genes encoding "contractile" proteins, such as SMα-actin (ACTA2), SM-MHC (myosin-11) and SM22α (transgelin). When exposed to various pathological conditions (e.g., pro-atherogenic risk factors, hypertension), SMC undergo phenotypic modulation, a bioprocess enabling SMC to de-differentiate in immature stages or trans-differentiate into other cell phenotypes. As recent studies suggest, the process of SMC phenotypic transformation involves five distinct states characterized by different patterns of cell growth, differentiation, migration, matrix protein expression and declined contractility. These changes are mediated via the action of several transcriptional regulators, including myocardin and serum response factor. Conversely, other factors, including Kruppel-like factor 4 and nuclear factor-κB, can inhibit SMC differentiation and growth arrest, while factors such as yin yang-1, can promote SMC differentiation whilst inhibiting proliferation. This article reviews recent advances in our understanding of regulatory mechanisms governing SMC phenotypic modulation. We propose the concept that transcription factors mediating this switching are important biomarkers and potential pharmacological targets for therapeutic intervention in cardiovascular disease.

Guidelines for the design and conduct of human clinical trials on ingestion-time differences - chronopharmacology and chronotherapy - of hypertension medications.

Current hypertension guidelines fail to provide a recommendation on when-to-treat, thus disregarding relevant circadian rhythms that regulate blood pressure (BP) level and 24 h patterning and medication pharmacokinetics and pharmacodynamics. The ideal purpose of ingestion-time (chronopharmacology, i.e. biological rhythm-dependent effects on the kinetics and dynamics of medications, and chronotherapy, i.e. the timing of pharmaceutical and other treatments to optimize efficacy and safety) trials should be to explore the potential impact of endogenous circadian rhythms on the effects of medications. Such investigations and outcome trials mandate adherence to the basic standards of human chronobiology research. In-depth review of the more than 150 human hypertension pharmacology and therapeutic trials published since 1974 that address the differential impact of upon-waking/morning versus at-bedtime/evening schedule of treatment reveals diverse protocols of sometimes suboptimal or defective design and conduct. Many have been "time-of-day," i.e. morning versus evening, rather than circadian-time-based, and some relied on wake-time office BP rather than around-the-clock ambulatory BP measurements (ABPM). Additionally, most past studies have been of too small sample size and thus statistically underpowered. As of yet, there has been no consensual agreement on the proper design, methods and conduct of such trials. This Position Statement recommends ingestion-time hypertension trials to follow minimum guidelines: (i) Recruitment of participants should be restricted to hypertensive individuals diagnosed according to ABPM diagnostic thresholds and of a comparable activity/sleep routine. (ii) Tested treatment-times should be selected according to internal biological time, expressed by the awakening and bed times of the sleep/wake cycle. (iii) ABPM should be the primary or sole method of BP assessment. (iv) The minimum-required features for analysis of the ABPM-determined 24 h BP pattern ought to be the asleep (not "nighttime") BP mean and sleep-time relative BP decline, calculated in reference to the activity/rest cycle per individual. (v) ABPM-obtained BP means should be derived by the so-called adjusted calculation procedure, not by inaccurate arithmetic averages. (vi) ABPM should be performed with validated and calibrated devices at least hourly throughout two or more consecutive 24 h periods (48 h in total) to achieve the highest reproducibility of mean wake-time, sleep-time and 48 h BP values plus the reliable classification of dipping status. (vii) Calculation of minimum required sample size in adherence with proper statistical methods must be provided. (viii) Hypertension chronopharmacology and chronotherapy trials should preferably be randomized double-blind, randomized open-label with blinded-endpoint, or crossover in design, the latter with sufficient washout period between tested treatment-time regimens.

Chronotherapy of cardiac and vascular disease: timing medications to circadian rhythms to optimize treatment effects and outcomes.

Circadian rhythms impact cardiac and vascular pathophysiology, resulting in 24-hour patterning of symptoms and life-threatening/ending events (chronopathology), plus kinetics and dynamics of medications (chronopharmacology), resulting in administration-time differences in efficacy and safety. Scheduling medications according to circadian rhythm determinants (chronotherapy) can improve treatment effects, for example, before dinner/bedtime ingestion of cholesterol-lowering medications and acetylsalicylic acid, respectively, exerts enhanced control of hypercholesterolemia and after-awakening peak of platelet aggregation; bedtime ingestion of conventional hypertension medications optimizes normalization of sleep-time blood pressure (BP)-strongest independent BP marker of cardiovascular disease (CVD) risk-and most effectively prevents (chronoprevention) CVD morbidity and mortality. Exploration of chronotherapeutic strategies to improve management of cardiac arrhythmias and vascular pathophysiology is still awaited.

Tongxinluo Improves Apolipoprotein E-Deficient Mouse Heart Function.

BACKGROUND: Our previous studies have shown that Tongxinluo (TXL), a compound Chinese medicine, can decrease myocardial ischemia-reperfusion injury, protect capillary endothelium function, and lessen cardiac ventricle reconstitution in animal models. The aim of this study was to illuminate whether TXL can improve hypercholesterolemia-impaired heart function by protecting artery endothelial function and increasing microvascular density (MVD) in heart. Furthermore, we will explore the underlying molecular mechanism of TXL cardiovascular protection. METHODS: After intragastric administration of TXL (0.1 ml/10 g body weight) to C57BL/6J wild-type mice (n = 8) and ApoE-/- mice (n = 8), total cholesterol, high-density lipoprotein-cholesterol, very-low-density lipoprotein (VLDL)-cholesterol, triglyceride, and blood glucose levels in serum were measured. The parameters of heart rate (HR), left ventricular diastolic end diameter, and left ventricular systolic end diameter were harvested by ultrasonic cardiogram. The left ventricular ejection fraction, stroke volume, cardiac output, and left ventricular fractional shortening were calculated. Meanwhile, aorta peak systolic flow velocity (PSV), end diastolic flow velocity, and mean flow velocity (MFV) were measured. The pulsatility index (PI) and resistant index were calculated in order to evaluate the vascular elasticity and resistance. The endothelium-dependent vasodilatation was evaluated by relaxation of aortic rings in response to acetylcholine. Western blotting and real-time quantitative reverse transcription polymerase chain reaction were performed for protein and gene analyses of vascular endothelial growth factor (VEGF). Immunohistochemical detection was performed for myocardial CD34 expression. Data in this study were compared by one-way analysis of variance between groups. A value of P < 0.05 was considered statistically significant. RESULTS: Although there was no significant decrease of cholesterol level (F = 2.300, P = 0.240), TXL inhibited the level of triglyceride and VLDL (F = 9.209, P = 0.024 and F = 9.786, P = 0.020, respectively) in ApoE-/- mice. TXL improved heart function of ApoE-/- mice owing to the elevations of LVEF, SV, CO, and LVFS (all P < 0.05). TXL enhanced aortic PSV and MFV (F = 10.774, P = 0.024 and F = 11.354, P = 0.020, respectively) and reduced PI of ApoE-/- mice (1.41 ± 0.17 vs. 1.60 ± 0.17; P = 0.037). After incubation with 10 µmol/L acetylcholine, the ApoE-/- mice treated with TXL aortic segment relaxed by 44% ± 3%, significantly higher than control group mice (F = 9.280, P = 0.040). TXL also restrain the angiogenesis of ApoE-/- mice aorta (F = 21.223, P = 0.010). Compared with C57BL/6J mice, the MVD was decreased in heart tissue of untreated ApoE-/- mice (54.0 ± 3.0/mm2 vs. 75.0 ± 2.0/mm2; F = 16.054, P = 0.010). However, TXL could significantly enhance MVD (65.0 ± 5.0/mm2 vs. 54.0 ± 3.0/mm2; F = 11.929, P = 0.020) in treated ApoE-/- mice. In addition, TXL obviously increased the expression of VEGF protein determined by Western blot (F = 20.247, P = 0.004). CONCLUSIONS: TXL obviously improves the ApoE-/- mouse heart function from different pathways, including reduces blood fat to lessen atherosclerosis; enhances aortic impulsivity, blood supply capacity, and vessel elasticity; improves endothelium-dependent vasodilatation; restraines angiogenesis of aorta-contained plaque; and enhances MVD of heart. The molecular mechanism of MVD enhancement maybe relate with increased VEGF expression.

Omega-3 fatty acids attenuate constitutive and insulin-induced CD36 expression through a suppression of PPAR α/γ activity in microvascular endothelial cells.

Microvascular dysfunction occurs in insulin resistance and/or hyperinsulinaemia. Enhanced uptake of free fatty acids (FFA) and oxidised low-density lipoproteins (oxLDL) may lead to oxidative stress and microvascular dysfunction interacting with CD36, a PPARα/γ-regulated scavenger receptor and long-chain FFA transporter. We investigated CD36 expression and CD36-mediated oxLDL uptake before and after insulin treatment in human dermal microvascular endothelial cells (HMVECs), ± different types of fatty acids (FA), including palmitic, oleic, linoleic, arachidonic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. Insulin (10(-8) and 10(-7) M) time-dependently increased DiI-oxLDL uptake and CD36 surface expression (by 30 ± 13%, p<0.05 vs. untreated control after 24 hours incubation), as assessed by ELISA and flow cytometry, an effect that was potentiated by the PI3-kinase inhibitor wortmannin and reverted by the ERK1/2 inhibitor PD98059 and the PPARα/γ antagonist GW9662. A ≥ 24 hour exposure to 50 µM DHA or EPA, but not other FA, blunted both the constitutive (by 23 ± 3% and 29 ± 2%, respectively, p<0.05 for both) and insulin-induced CD36 expressions (by 45 ± 27 % and 12 ± 3 %, respectively, p<0.05 for both), along with insulin-induced uptake of DiI-oxLDL and the downregulation of phosphorylated endothelial nitric oxide synthase (P-eNOS). At gel shift assays, DHA reverted insulin-induced basal and oxLDL-stimulated transactivation of PPRE and DNA binding of PPARα/γ and NF-κB. In conclusion, omega-3 fatty acids blunt the increased CD36 expression and activity promoted by high concentrations of insulin. Such mechanisms may be the basis for the use of omega-3 fatty acids in diabetic microvasculopathy.

Tongxinluo reduces myocardial no-reflow and ischemia-reperfusion injury by stimulating the phosphorylation of eNOS via the PKA pathway.

The objective of the present study was to investigate whether pretreatment with single low loading dose of tongxinluo (TXL), a traditional Chinese medicine, 1 h before myocardial ischemia could attenuate no-reflow and ischemia-reperfusion injury by regulating endothelial nitric oxide synthase (eNOS) via the PKA pathway. In a 90-min ischemia and 3-h reperfusion model, minipigs were randomly assigned to the following groups: sham, control, TXL (0.05 g/kg, gavaged 1 h before ischemia), TXL + H-89 (a PKA inhibitor, intravenously infused at a dose of 1.0 µg·kg(-1)·min(-1) 30 min before ischemia), and TXL + N(ω)-nitro-L-arginine (L-NNA; an eNOS inhibitor, intravenously administered at a dose of 10 mg/kg 30 min before ischemia). TXL decreased creatine kinase (CK) activity (P < 0.05) and reduced the no-reflow area from 48.6% to 9.5% and infarct size from 78.5% to 59.2% (P < 0.05), whereas these effects of TXL were partially abolished by H-89 and completely reversed by L-NNA. TXL elevated PKA activity and the expression of PKA, Thr(198) phosphorylated PKA, Ser(1179) phosphorylated eNOS, and Ser(635) phosphorylated eNOS in the ischemic myocardium. H-89 repressed the TXL-induced enhancement of PKA activity and phosphorylation of eNOS at Ser(635), and L-NNA counteracted the phosphorylation of eNOS at Ser(1179) and Ser(635) without an apparent influence on PKA activity. In conclusion, pretreatment with a single low loading dose of TXL 1 h before ischemia reduces myocardial no-reflow and ischemia-reperfusion injury by upregulating the phosphorylation of eNOS at Ser(1179) and Ser(635), and this effect is partially mediated by the PKA pathway.

Nitric oxide bioactivity of traditional Chinese medicines used for cardiovascular indications.

Traditional Chinese medicine (TCM) has been used for centuries to treat and prevent certain ailments and diseases. Although TCM has served as mainstream medical care throughout Asia for many generations, it is considered an alternative medical system in much of the Western world. Because many TCMs are used primarily for cardiovascular indications characterized by a nitric oxide (NO) insufficiency, we hypothesized that some, if not all, of these TCMs have a robust NO bioactivity that may act to restore NO homeostasis. We tested a group of convenience samples of TCMs obtained in the United States for endogenous nitrite, nitrate, nitroso, and nitrite reductase activity as well as their ability to relax isolated aortic rings. The results from this study reveal that all of the TCMs tested reveal NO bioactivity through their inherent nitrite and nitrate content and their ability to reduce nitrite to NO. Many of the TCM extracts contain a nitrite reductase activity greater by 1000 times that of biological tissues. Repletion of biological nitrite and nitrate by these extracts and providing a natural system for NO generation in both endothelium-dependent and -independent mechanisms may account for some of the therapeutic effects of TCMs.