Circadian Rhythms of Risk Factors and Management in Atherosclerotic and Hypertensive Vascular Disease: Modern Chronobiological Perspectives of an Ancient Disease

Abstract

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 shiftwork, 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.

INTRODUCTION

Atherosclerosis is a chronic, inflammatory disease of the arteries that gives rise to two life-threatening complications, myocardial infarction and cerebral stroke. The pathogenesis of atherosclerosis is characterized by cholesterol deposition, smooth muscle proliferation, inflammatory cell infiltration, and connective tissue accumulation, which manifests as plaques (atheromas) in the tunica intima of the arterial wall. When the plaque narrows the arterial lumen, which is easily discernable by X-ray radiography, blood flow becomes restricted and causes distal tissue ischemia, and when it fully occludes the lumen, blood flow is interrupted and may cause a non-fatal or fatal coronary or cerebral vascular event. Atherosclerosis-associated coronary and cerebral diseases are a leading cause of death not only in the United States (https://www.cdc.gov/chronicdisease/resources/publications/factsheets/heart-disease-stroke.htm) but worldwide (https://www.who.int/health-topics/cardiovascular-diseases#tab=tab_1), even today during the COVID-19 pandemic. As illustrated in Figure 1, the predisposing, causative, and conditional risk factors of atherosclerosis include, based upon their circadian patterns, two subgroups: (i) Oscillatable risk factors that entail circadian clock-dependent activities, such as unhealthy lifestyle (such as sedentary habit, poor diet, tobacco smoking, excessive alcohol consumption, emotional stress), metabolic dysfunction (such as, insulin resistance/type 2 diabetes, obesity, dyslipidemia (such as elevated low density lipoprotein and diminished high density lipoprotein), hypertension, obstructive sleep apnea (OSA), and shiftwork; and (2) Non-oscillatable risk factors, such as age, male sex, family history, genetic defects, and pandemics. These factors may act individually or collectively with influence of circadian rhythms to activate leukocytes or immune cells to release cytokines that induce programmed cell death (apoptosis) of vascular cells and trigger vascular injury, tissue remodeling, and plaque formation (Geng and Libby 2002). Atherosclerotic and/or hypertensive vascular injury is causal of various pathologic conditions, ranging from asymptomatic intima hyperplasia to symptomatic peripheral arterial disease and acute arterial occlusion. Rupture or disruption of the atherosclerotic plaque structure initiates a thrombogenic cascade entailing the activation of platelet aggregation, coagulation factors, and inflammatory cell adhesion, and consequently ischemic injury of distal tissues or organs due to significant occlusion of the affected artery. When the rupture occurs in a plaque situated in a coronary artery, the result can be an acute myocardial infarction (AMI) or sudden coronary death, and when it occurs in a cerebral artery, the result can be an ischemic or hemorrhagic stroke (IS, HS).

Figure 1. Circadian oscillating and non-oscillating risk factors for atherosclerosis and its coronary and cerebral complications.

Various metabolic and functional processes are in association with risk factors, with or without circadian oscillation, which promote the development of atherosclerotic and hypertensive vascular disease and its life-fatal complications, namely coronary and cerebral ischemia and infarction.

Cardiovascular (CVD) events in most people occur more often during the initial hours of wakefulness than other time during the 24 h (Muller 1999; Muller et al. 1989); however, such events in those with OSA occur more often during sleep (Kuniyoshi et al. 2008). As later discussed, the upon-awakening excess of CVD events in the former group results in large part from circadian clock-driven rhythms in blood coagulation, vascular physiology, and pathophysiology, while the excess in CVD events during sleep in the latter group results from pathophysiologic stresses induced by increased ventilatory and cardiac effort.

Many molecular oscillators or clock gene products or proteins have been identified in the mammalian circadian system that play a critical role in the generation and maintenance of cell-autonomous rhythms (Dibner et al. 2010). For instance, the Circadian Locomotor Output Cycle Kaput (CLOCK) and Brain Muscle Aryl Hydrocarbon Receptor Nuclear Translocator-Like 1 (BMAL1, encoded by the ARNTL gene) are two clock proteins that act as primary regulators of circadian rhythms. In this review, we will update the progress of circadian gene research related to atherosclerosis, hypertension, and other metabolic disorders. Finally, we will summarize recent data from several clinical studies using chronotherapeutic approaches that demonstrate progress in the mitigation of risk for morbid and mortal cardiac and vascular outcomes.

ATHEROSCLEROSIS: MODERN OR OLD DISEASE OF CIVILIZATION

The histological properties and inflammatory characteristics of arterial plaque formation, calcification, and ossification were initially described in 1848 by the renowned German pathologist Rudolf Virchow (Virchow 1989). Atherosclerosis is viewed as a chronic disease of “modern” times, i.e., of developed societies and countries, such as the United States and Europe. Indeed, the prevalence of atherosclerosis and its complications, e.g., AMI, IS, and HS, are higher in developed than in developing societies, even though during the past few decades the incidence of coronary artery disease (CAD) has been gradually declining with the elaboration and application of increasingly effective therapeutic and preventive management strategies (Herrington et al. 2016). Elevated atherosclerotic-associated coronary and cerebral arterial morbidity and mortality are also prevalent in developing countries undergoing rapid economic growth, such as China and India. Atherosclerosis is often undiagnosed until clinical symptoms present or a cardio/cerebrovascular event occurs. Therefore, the prevalence of subclinical atherosclerosis may be underestimated. Nonetheless, life-threatening complications of advance atherosclerosis, namely myocardial and cerebral infarction, AMI, IS, and HS, appear to be higher in developed than in developing societies.

Medical examination of well-preserved human remains of different countries and cultures indicates atherosclerosis is in fact not a “modern” but an “ancient” disease of civilizations. Morphologically, the pattern of atherosclerotic plaque formation in well-preserved remains of ancient humans is similar to that of humans today. Reyman et al. performed histopathological and radiological studies of Egyptian mummies dating between 3000 and 2000 BCE, revealing atherosclerotic lesions in the aorta (Reyman et al. 1976). Thompson et al. performed whole body computer tomography (CT) on 137 ancient Egyptian, Peruvian, Puebloan (southwest America), and Unangan (Aleutian Islands) mummies, discovering clearly illustrated calcified plaques in the wall of different arteries (Thompson et al. 2013). Collectively, these findings confirm the initial report in 1931 by Long et al. of autopsy-established coronary and renal atherosclerosis of Lady Teye, an ancient Egyptian woman who died at approximately 50 years of age (Long et al. 1931). Additionally, Allam et al. reported strong similarities in the demographic characteristics and presence and extent of vascular calcification of atherosclerotic lesions determined by whole-body CT scans among 178 modern and 76 ancient (mummified) Egyptians dating to between 3100 BCE and 364 CE (Allam et al. 2014). Like modern Egyptians, ancient Egyptians displayed calcified plaques in the aortoiliac wall almost a decade earlier than in event-related, e.g., coronary and carotid, arteries, suggesting a common age-dependent process of atherosclerotic lesion buildup in both ancient and modern Egyptians. Furthermore, existence of angina pectoris or CAD has been documented in mummified remains of ancient Egyptians (Hajar 2017). However, pathophysiologic and epidemiologic evidence is lacking as to the prevalence of atherosclerosis and heart disease in ancient populations. The autopsy and CT scans of ancient human remains substantiate the existence of atherosclerosis in people living several thousand years ago, a finding that has important implications for the health of people today, irrespective of racial, geographic, lifestyle, socioeconomic, cultural, and other differences.

CHRONOGENOMICS OF ATHEROSCLEROSIS

Genomic and other panomic profiling data have identified genetic factors possibly associated with vascular pathologies that may be modulated by circadian rhythms. Moreover, circadian rhythms in so-termed interactome networks can, if established appropriately, help dissect temporal relationships between genes or proteins that may have broad applicability to the chronopathology (biological rhythms in disease mechanisms and symptom manifestation) and chronotherapy (optimization of desired effects and/or minimization or aversion of adverse effects of medications though timing to biological rhythms) across a broad spectrum of human health disorders. Therefore, such information constitutes an unbiased source, when interpreted in terms of circadian time, not only to comprehensively understand biologically relevant activities of human diseases but interactions of medications at the cell, organ, and whole body level that conceptually comprise the chronopharmacogenomics of single medications as well as drug-drug interactions of multiple pharmacotherapies (Geng et al. 2021). Assessment of the activity of this conceptualized chronopharmacointeractome can also help provide more in-depth understanding of the mechanisms of medical conditions and diseases, such as atherosclerosis and arterial hypertension, because it enables simultaneous consideration of multiple relevant genes, metabolites, and proteins that may interact in a circadian-time-dependent manner during their development and overt manifestation.

Genome-wide-association studies (GWAS) and polygenic analyses are important methods used to identify genomic regions that contribute to the pathogenesis of atherosclerotic vascular disease (Agbaedeng et al. 2021). GWAS have been able to identify >100 single nucleotide polymorphisms (SNPs) that affect variations of cardiovascular functions, such as metabolic profiles and blood pressure (Kessler et al. 2021). However, GWAS findings need to be integrated with phenotypic data to generate Quantitative Trait Loci (eQTL). Recent investigations reveal about 30% of mammalian genes controlled by eQTLs strongly contribute to the risk for developing complex diseases and adverse drug-drug interactions. For example, several independent GWAS propose certain SNPs in chromosome 9p21 are related to heart diseases (Kessler et al. 2021). However, these SNPs are localized in the non-coding region of the chromosome, and the nearest genes are >100 Kb away. Therefore, the causality of SNPs with regard to atherosclerosis and pathological vascular outcomes is uncertain. GWAS and other genetic studies have documented that circadian bioclocks are endogenously regulated by various gene products functional as oscillators that control 24 h physiological and behavioral processes (Rijo-Ferreira and Takahashi 2019). The central circadian clock exerts control over multiple aspects of diverse pathological conditions, including atherosclerosis, hypertension, and other metabolic disorders. It is important to unravel the circadian clock’s role in controlling cardiovascular conditions. Chronogenomics provide opportunities for the discovery of new and improved prevention and treatment strategies for cardiovascular diseases.

MAMMALIAN CIRCADIAN MOLECULAR CLOCK

The mammalian circadian system is regulated by a group of molecular oscillators or clock proteins critical for the generation and maintenance of cell-autonomous rhythms (Dibner et al. 2010). The Circadian Locomotor Output Cycle Kaput (CLOCK) and Brain Muscle Aryl Hydrocarbon Receptor Nuclear Translocator-Like 1 (BMAL1, encoded by the ARNTL gene) are two clock proteins that act as primary regulators (Figure 2). Both belong to the basic helix-loop-helix–PER-ARNT-SIM (bHLH–PAS) transcription factor family that activates transcription of target genes by forming heterodimers and binding to E-box/enhancer elements (5′-CACGTG-3′) in promoter/enhancer regions. As a member of the bHLH–PAS protein family, neuronal PAS 2 (NPAS2) forms heterodimers with BMAL1 and subsequently controls E-box element-dependent gene transcription (Asher and Schibler 2006). The targets include proteins, such as PERIODs (PERs: PER1, PER2, and PER3) and CRYPTOCHROMEs (CRYs: CRY1 and CRY2), that form a negative feedback loop. Accumulated PER and CRY proteins form repressive complexes that suppress E-box-mediated transcription by binding to CLOCK/BMAL1 heterodimers, whereas PER and CRY degradation terminates this repression and reinitiates transcription (Gekakis et al. 1998; Hogenesch et al. 1998; Kume et al. 1999; Shearman et al. 2000). The CLOCK/BMAL1-initiated loop serves as the key mammalian clock unit. A secondary loop consists of sets of circadian nuclear receptors, in particular, REV-ERBs (REV-ERBα and β, respectively encoded by NR1D1 and NR1D2) and retinoic acid receptor-related orphan nuclear receptors (RORs: RORα-γ) that are also under transcriptional control of the CLOCK:BMAL1 heterodimer (Figure 2). Curtis et al. (2007) reported the circadian variations in regulation of blood pressure (BP) and the vascular response to asynchronous stress in mice, in which the core clock genes of Bmal1(-/-), Clock(mut), and Npas2(mut) are deleted or mutated. The clock gene variants influence the time-dependent incidence of cardiovascular events by controlling the integration of selective asynchronous stress responses with an underlying circadian rhythm in cardiovascular function.

Figure 2. Circadian rhythmic regulation of clock gene expression and modulation of circadian variations of risk factors for the pathogenesis of atherosclerotic and hypertensive vascular disease.

In engagement with the circadian receptor RORα, BMAL1 and CLOCK form a heterodimeric complex to positively regulate expression of target genes coding for PERs and CRYs, two factors that repress the transcription activity of BMAL1/CLOCK complex, as part of the negative feedback loop of circadian rhythm. Generated at high levels during atherogenesis, oxidative products of cholesterol or oxysterols bind to RORα proteins, contributing to the transcriptional control of the Bmal1 and Clock genes. The circadian clock gene changes underlie the development of adverse events in atherosclerotic and hypertensive vascular disease.

REV-ERBs and RORs compete to occupy RORs/REV-ERBs-responsive elements (RREs) located in the promoter/enhancer regions of their target genes. RORs usually activate RRE-mediated transcription, whereas REV-ERBs strongly suppress it (Preitner et al. 2002; Sato et al. 2004; Ueda et al. 2002). This stabilizing loop was originally considered as accessory, because only moderate phenotypes were observed in mutant mice bearing null alleles of any of these genes. However, subsequent studies utilizing inducible double knockouts for both NR1D1 and NR1D2 revealed that their compensatory activity yielded these subtle phenotypes, and that REV-ERBs are required for normal circadian period regulation (Cho et al. 2012). REV-ERBs also control circadian outputs by cooperating with cell type specific transcriptional regulators (Chung et al. 2014; Zhang et al. 2015). Additional feedback loops involving proline and acidic amino acid-rich basic leucine zipper proteins (PARbZip), such as D-box binding protein (DBP) and E4 promoter-binding protein 4 (E4BP4), plus several members of the bHLH transcription family (BHLHE40 and BHLHE41) also intersect with the main loops to further regulate and mediate circadian expression of subsets of clock-controlled genes (Honma et al. 2002; Mitsui et al. 2001). The circadian nuclear receptors REV-ERBs and RORs mediate many physiological processes, such as regulation of circadian rhythms, development, metabolism, immunity, and brain functioning. Members of the nuclear receptor superfamily are ligand-activated transcription factors that act as intracellular receptors for cell-permeable ligands. Recent studies have discovered native, endogenous ligands (e.g., cholesterol oxides or oxysterols) for these circadian nuclear receptors, thereby encouraging development of synthetic ligands for therapeutic application to manage circadian rhythm-related diseases (Kojetin and Burris 2014). Changes in clock gene expression may contribute to the pathogenesis of atherosclerosis. Aberrant circadian rhythms, increased pathological remodeling, vascular endothelial dysfunction, as well as attenuation of Akt and nitric oxide signaling have been found in the arteries of mice with Bmal1-deficiency and Clock mutation (Anea et al. 2009). Bmal1 deficiency induces oxidative stress, inflammation, and atherosclerosis in ApoE-null mice (Xie et al. 2020). Furthermore, human carotid plaque-derived smooth muscle cells display circadian patterns of Bmal1, Cry1, Cry2, Per1, Per2, Per3, and Rev-erb-α mRNA that differ from those of normal carotid arterial tissue (Lin, et al. 2014).

Although REV-ERBs were initially identified as orphan nuclear receptors, subsequent studies revealed heme binds to their ligand binding domain (LBD) (Raghuram et al. 2007; Yin et al. 2007). The discovery of endogenous REV-ERBs ligands led to the identification of chemical scaffolds that can act as synthetic ligands. The first synthetic REV-ERB ligand was GSK4112 (Meng et al. 2008). It enhances recruitment of nuclear receptor co-repressor (NCoR) and histone deacetylase 3 (HDAC3) to their target promoters to then repress target gene transcription (Grant et al. 2010). While GSK4112 does not exhibit favorable pharmacokinetics, it has paved the way for subsequent development of other synthetic REV-ERBs ligands. To improve pharmacokinetics, potency, and efficacy, Burris developed additional REV-ERB agonists, such as SR9009 and SR9011, that have been found to be more suitable for in vivo applications (Burris 2008). Both demonstrated the therapeutic efficacy of small molecule REV-ERB modulators in the treatment of circadian rhythm-related metabolic and sleep disorders (Solt et al. 2012). Although several REV-ERBs agonists are now known, SR8278 is the only antagonist so far identified. It inhibits the transcriptional repression activity of REV-ERBs, thereby enhancing RRE-mediated transcription (Kojetin et al. 2011). Although the in vivo applications of SR8278 have thus far been limited, it constitutes a convenient tool to temporally inhibit REV-ERB activity in target cells or tissues. Dierickx, et al. (2019) have recently demonstrated that SR9009 exerts REV-ERB-independent effects on cell proliferation and metabolism. Thus, the exact role of REV-ERB defined by SR9009 or similar agents in regulating circadian rhythmicity of metabolism and function remains to be determined.

During the development of atherosclerosis, cholesterol is converted by oxidation into oxysterols, which behave as high-affinity endogenous ROR modulators or ligands. Oxysterol ligands bind directly to the RORα/γ LBD and act as inverse agonists by modulating interaction of co-regulators. RORs are evolutionarily related to retinoic acid receptors, which are regulated by circadian rhythms or the day-night or light-dark 24h cycle, and mediate a broad range of nuclear factor activation or suppression, particularly via BMAL1-CLOCK interaction (Figure 2). Interestingly, all-trans retinoic acids recognize the LBD of RORβ, but not the LBD of RORα/γ, suggesting subtype specificity. The first synthetic ligand and inverse agonist identified for RORα/γ is the liver X receptor agonist T0901317 (Kumar et al. 2010). Subsequently, a series of RORα/γ agonists or inverse agonists were developed, as reviewed in detail elsewhere (Kojetin and Burris 2014). In recent years, Chen et al. identified the natural polymethoxylated flavone nobiletin to be an enhancer of the circadian amplitude of molecular rhythms by acting on RORs (Chen et al. 2012; He et al. 2016). The cluster of differentiation-1d (CD1d) recognizes and internalizes pro-atherogenic lipids, such as oxysterols, which subsequently activate nuclear peroxisome proliferator-activated receptor-γ (PPAR-γ) (Rosales et al. 2015). PPAR-γ is involved in CLOCK:BMAL1 chromatin recruitment and cyclic activation of surrogate pathways in response to nutrient challenges (Eckel-Mahan 2013).

CIRCADIAN REGULATION OF VASCULAR PROGENITOR CELL FUNCTION

Cardiovascular tissue injury by atherosclerosis triggers in response tissue repair and regeneration. Stem cell malfunction reduces the capacity of tissue repair and regeneration, and thereby contributes to plaque necrotic core formation and enlargement. Circadian rhythms play an important role in the regulation of stem cell growth, metabolism, and function. The circadian clock exerts regulatory impacts on the homeostasis and functioning of stem cell metabolism and function in cardiovascular tissues. Specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes mellitus (Bhatwadekar et al. 2017). In two Bmal1fx/fx;Tek-Cre mouse models -- a retinal ischemia/reperfusion model and a neointimal hyperplasia femoral artery model -- the number of acellular capillaries in retinas was increased three-fold and nitrotyrosine staining one and half-fold. The mice also exhibited evidence of bone marrow denervation, demonstrating loss of neurofilament 200 staining. Moreover, injury of the femoral arteries resulted in 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Circulating CD34⁺/VEGFR2⁺ (vascular endothelial growth factor receptor 2⁺) endothelial progenitor cells (EPCs) play a central role in endothelial homeostasis and vascular repair. Reduced number and functional activity of EPCs are associated with several CVD risk factors. Kim et al. demonstrated the relationship between the BP circadian rhythm and number of EPCs in 45 essential hypertension patients diagnosed by 24 h ambulatory BP monitoring (ABPM) (Kim et al. 2012). The number of circulating EPCs was significantly reduced in patients exhibiting the abnormal non-dipper (mean nocturnal SBP decline <10% of mean daytime SBP) as compared to those exhibiting the normative dipper [mean nocturnal SBP decline ≥10 but ≤20% of mean daytime SBP]) phenotype. Moreover, the number of circulating EPCs correlated positively with circadian changes in systolic (S) and diastolic (D) BP (SBP and DBP). It is noteworthy the nocturnal hypoxemia of obstructive sleep apnea (OSA) is associated with increased endothelial cell number (CD34⁺), which may promote vascular repair (Lui et al. 2013). However, accumulation of OSA-caused arterial gas embolisms may lead to diminution over time in the number of early EPCs (CD133⁺) and endothelial repair capacity, thus contributing to vascular pathogenesis.

Wang et al. reported dysfunction of endothelial cells and EPCs plus increased vascular senescence in mice with the circadian gene Per² mutant, Per²(m/m) (Wang et al. 2008). Endothelial cells of Per²(m/m) mice exhibited increased protein kinase AKT signaling, enhanced senescence, and impaired vascular network formation and proliferation. Additionally, Per²(m/m) mice evidenced impaired blood flow recovery and auto-amputation of the distal limb when subjected to experimental hind-limb ischemia. Furthermore, implantation of matrigel, a basement membrane matrix utilized for cell culturing, into Per²(m/m) mice revealed poor neovascularization capacity of EPCs. The number of basal EPCs of wild-type and Per²(m/m) mice was similar; however, compared with wild-type bone-marrow-transplanted mice, mobilization of EPCs was impaired in Per²(m/m) bone-marrow-transplanted mice in response to ischemia or vascular endothelial growth factor (VEGF) stimulation. Finally, bone marrow transplantation or infusion of wild-type EPCs restored blood flow recovery and prevented auto-amputation in Per²(m/m) mice.

CIRCADIAN REGULATION OF VASCULAR INFLAMMATION

Many proatherogenic factors exhibit prominent circadian variation, including inflammatory cells (e.g., lymphocytes and macrophages), cytokines, and lipids (e.g., ox-LDL). These factors contribute to the pathogenesis of atherosclerosis and are regulated in a chronobiologic fashion. De Juan et al. (2019) through an in vivo investigation found that artery-associated sympathetic innervation drives rhythmic vascular inflammation of murine arteries and veins. They observed utilizing real-time multichannel fluorescence intravital microscopy that leukocytes were recruitted to the arterial and venous macrovasculature and microvasculature in a tumor necrosis factor-α-induced acute inflammation mouse model. This was complemented with pharmacological ablation of the sympathetic nerves or adrenergic receptors to assess their relevance for the circadian rhythm of leukocyte adhesion. Additionally, the investigators genetically targeted the key circadian clock gene Bmal1 in a lineage-specific manner to dissect the importance of the 24 h oscillations in leukocytes and components of the vessel wall in this process. In vivo quantitative imaging analyses revealed circadian rhythmicity in leukocyte recruitment and adherence to arteries and veins of the mouse macrovasculature and microvasculature. However, there was an about 12 h disparity in the phasing of the rhythm of the arteries versus veins in the expression of pro-migratory molecules that is governed by the different rhythmic microenvironment and oscillatory pattern of the specific blood vessel type. Furthermore, loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. BMAL1 deletion may lead to a proinflammatory state, which accelerates atherosclerosis, at least as shown in murine models (Yang et al. 2020). Leukocyte adhesion in arteries occurs at highest rates early during the animals’ daily rest span, and at lowest rates around the middle of their daily activity span, the consequence being predisposition to acute thrombosis that differs according to circadian time. In contrast, leukocyte adhesion in veins is lowest early during the animals’ daily rest span and greatest during the middle of their activity span. These findings point to an important and previously unrecognized role of blood vessel-associated sympathetic nerve activities in governing the circadian rhythmicity of vascular inflammation in arteries and veins. Furthermore, these findings provide new insight concerning the known temporal patterning during the 24 h of vessel type-specific thrombotic events, i.e., AMI, IS, and HS (Muller 1999; Muller et al. 1989).

CIRCADIAN RHYTHM OF ATHEROTHROMBOSIS

The integrity of blood vessels both in normal and pathophysiological conditions (e.g., atherosclerosis and hypertension) is maintained through balance between anticoagulant and procoagulant processes. As previously reviewed in detail (Haus 2007), many of these processes are circadian rhythmic. The activities of the endothelium of importance to hemostasis following vascular injury are vasoconstriction provoked by endothelin, anticoagulation stimulated by heparin and thrombomodulin, vasodilation and anti-platelet effects mediated by nitric oxide (NO) and prostaglandins, and fibrinolysis generated by tissue-type activator (t-Pa) and plasminogen activator inhibitor type 1 (PAI-1).

An initial important response to vascular injury is thrombosis. Under normal circumstances, platelet number displays a prominent 24 h pattern, with the nadir around waking time and peak late during wakefulness (Haus et al. 1990). Several elements of thrombosis and coagulation, such as platelet aggregation, ß-thromboglobulin, and platelet surface activated glycoprotein (GP), i.e., GPIIb-IIIa GPIb, and P-selectin concentration, are clearly circadian rhythmic. In vitro studies indicate platelet activation and aggregation are greatest around the time of the transition from sleep to wakefulness and least during the initial hours of sleep (Brezinski et al. 1988; Fujimura et al. 1992; Haus et al. 1990; Jafri et al. 1992; Scheer et al. 2011; Winther et al. 1992). Circadian rhythms of white blood cell (WBC) coagulation and free-radical formation (FRF) also are likely involved in thrombosis (Bridges et al. 1992a, 1992b). In men with stable ischemic heart disease, largest rise in WBC coagulation and FRF coincides with the peak of the circadian rhythm of platelet activity (Bridges et al. 1992b).

The coagulation cascade consists of a series of sequential complex chemical processes entailing many different plasma proteins, termed blood clotting or coagulation factors, which culminate in a blood clot barrier over the site of the vascular injury that enables healing (Mackman et al. 2007). The clotting process thus converts blood from a liquid to a solid at the site of injury. The coagulation cascade consists of so-called intrinsic, extrinsic, and common pathway factors, with the activity of many of them exhibiting circadian rhythmicity (Haus 2007; Haus et al. 1990). The intrinsic pathway, which involves Factors XII, XI, IX, and VIII, is triggered in response to internal blood vessel trauma. Clinical laboratory tests, like prothrombin time (PT), thromboplastin time (aPTT), and thrombin time (TT), utilized to assess the individual or combined activities of specific coagulation cascade factors, reveal significant circadian rhythmicity in blood clot formation (Haus et al. 1990; Petralito et al. 1982). Both PT, a measure of the activities of Factors VII, X, V, II, and fibrinogen that are members of both the extrinsic and common pathway, and aPTT, a measure of the activities of factors of the intrinsic and common pathways, i.e., Factors XII, XI, IX, VIII, X V, II, fibrinogen, prekallikrein, and high molecular weight kininogen, are shortest, indicative of blood hypercoagulability, around or following the transition daily from sleep to wakefulness (Haus 2007). In clinically healthy persons, plasma concentrations of markers of intravascular thrombin generation, i.e., prothrombin factors F1 and F2, also exhibit circadian variability with the peak time occurring after awakening from sleep (Kapiotis et al. 1997). In addition, D-dimer, an indicator of formed fibrin shows a similar circadian rhythm (Budkowska et al. 2019; Haus 2007; Kostovski et al. 2015). In clinically healthy men, both Protein C and S exhibit circadian rhythmicity, each showing a peak 6 h or so after awakening and nadir late during wakefulness or around bedtime. The anti-thrombosis (AT) process is also circadian rhythmic, but with opposite phasing, i.e., peak about 6 h before usual sleep onset and nadir early in the activity span (Ündar et al. 1999). Tissue factor (TF), which is expressed by perivascular cells, is the high-affinity receptor and cofactor for Factor VII; the TF/VII complex is the primary initiator of blood coagulation that acts to limit hemorrhage following blood vessel injury. Atherosclerotic plaques contain high levels of TF on macrophage foam cells and microvesicles that drive thrombus formation after plaque rupture (Grover and Mackman 2018). The inhibitor of the TF pathway, i.e., TF pathway inhibitor, exhibits circadian rhythmicity in clinically healthy men, with highest level found at the beginning of the daily activity span (Pinotti et al. 2005).

The fibrinolytic system, which is composed of plasmin, plasminogen activators, and their inhibitors, constitutes an important defense against intravascular thrombosis by complementing the effects of the various anticoagulant moieties, including Protein C, Protein S, antithrombin III, heparin cofactor II, and endothelial-derived platelet inhibitors NO and prostacyclin. Plasminogen is activated primarily by t-PA, and it is inhibited by plasminogen activator inhibitors (PAIs), the most important one being PAI-1 produced by endothelial cells of the vascular wall, liver, adipose tissue, and platelets (Haus 2007). The antigen concentration and activity of t-Pa and PAI-1 each exhibit a high-amplitude circadian rhythm (Andreotti and Kluft 1991; Andreotti et al. 1988; Angleton et al. 1989; Haus 2007). Highest t-Pa antigen concentration occurs around the commencement of wakefulness and lowest antigen t-Pa concentration occurs around bedtime or early sleep. However, the circadian rhythm in the activity of t-PA is differently phased; t-Pa activity is lowest during sleep and highest around the commencement of the wake span. The circadian rhythms in PAI-1 antigen concentration and activity, which are characterized by peak between mid-sleep and awakening and absolute nadir around the mid-to-late span of wakefulness, overrides the circadian rhythm in t-Pa activity and, therefore, is deterministic of the 24 h temporal variation in overall fibrinolytic activity (Andreotti and Kluft 1991; Andreotti et al. 1988; Angleton et al. 1989; Haus 2007; Scheer and Shea 2014).

The in vitro and in vivo synthesis of PAI-1 in vascular endothelium cells is driven by circadian clock components. Both CLOCK:BMAL1 and CLOCK:BMAL2 heterodimers activate the PAI promoter and make additional contributions to PAI-1 gene transcription (Chong et al. 2006; Schoenhard et al. 2003), with PER2 and PER3 exerting inhibitory activity (Oishi 2009; Oishi et al. 2009). Of particular importance is the finding that the integrity and peak activity of PAI-1 depend on the circadian rhythm of the renin-angiotensin-aldosterone system (RAAS) (Brown et al. 1998; Masuda et al. 2009). PAI-1 activity at the commencement of the activity span is exaggerated and extended by the staging of the circadian rhythm of the RAAS (as later discussed) and sympathetic nervous system, resulting in suppression of fibrinolysis at this biological time (Brown et al. 1998). Finally, it is important to recognize there is large inter-individual variation in PAI-1 antigen and activity expression that may result in difference between persons in the intensity of thrombogenesis. Experimental and clinical studies have identified polymorphisms (4G/5G alleles) in the promoter of the gene for PAI-1 that directly influences its circadian expression (Chong et al. 2006; Hoekstra et al. 2002). The extent of circadian variation in PAI-1 antigen concentration varies according to the genotype of the 4G/5G polymorphism in the promoter of the gene for PAI-1, with the 4G allele associated with highest post-awakening (morning in diurnally active humans) PAI-1 antigen expression (Hoekstra et al. 2002; van der Bom et al. 2003; White et al. 2015). The difference in PAI-1 antigen concentration is more pronounced among those homozygous for the 4G allele than among those of the other genotypes, but only during the post-awakening morning span (van der Bom et al. 2003). Additionally, the presence of obstructive sleep apnea (OSA), defined by an apnea-hypopnea index (HPI) ≥5 determined through overnight polysomnography, adversely affects circadian fibrinolytic balance, with higher PAI-1 activity and antigen level and significantly lower t-Pa activity in OSA versus non-OSA controls, which in the former may be predisposing for increased CVD risk (Bagai et al. 2014). These and other findings concerning the importance of the zygosity of the PAI-1 gene and medical co-morbidities indicate important inter-individual differences in the intensity and progression of thombogenesis and, thus, risk for atherothrombotic events (Gardemann et al. 1999; Haus 2007; Kumar et al. 2021), particularly during the initial hours of the human daily activity span (Muller 1999; Muller et al. 1989). Overall, the chronobiology of atherosclerosis reveals two distinguish, yet closely associated phases of atherogenesis, i.e., plaque formation and rupture. The clotting factors primarily contribute to the rhythms of ischemic events, such as vessel occlusion, whereas angiotensin II (Ang-II) and BP changes contribute towards plaque rupture.

RENIN-ANGIOTENSIN-ALDOSTEONE SYSTEM AND ATHEROGENESIS

The RAAS, which exhibits high-amplitude circadian rhythmicity as subsequently discussed, plays a critical role in the pathogenesis of vascular disease, primarily mediated through Ang-II (Mochel et al. 2015). Ang-II through its binding to angiotensin type 1 receptors (AT1Rs) of endothelial cells promotes endothelial dysfunction and inflammation and fibrosis of the tunica intima plus blood hypercoagulation and arterial BP elevation (Husain et al. 2015; Montezano et al. 2014; Poznyak et al. 2021; Silva et al. 2020). In the kidney, in response to arterial vasodilation, low blood sodium concentration, and ß₁-adrenoceptor stimulation, renin cleaves angiotensinogen into the inactive decapeptide angiotensin-I (Ang-I). Thereafter, Ang-I in circulating blood is converted into the octapeptide Ang-II, the most biologically active peptide of the RAAS through coupling to angiotensin type 1 and 2 receptors (AT1Rs and AT2Rs). Expression of AT2Rs is typically quite limited in adults, although it has been found to be increased following vascular injury to myocardial or renal tissue (Matavelli and Siragy 2015). Ang-II is also synthesized outside of the classical pathway in cells of different tissues (Dickson and Sigmund 2006; De Mello and Frohlich 2011; Kumar et al. 2012; Sparks et al. 2014). When bound to AT1Rs and AT2Rs in adjacent cells, Ang-II exerts actions in an autocrine/paracrine manner (Kumar et al. 2012) to augment effects induced or promoted by circulating Ang-II produced by the classical pathway. Local Ang-II formation is dependent on enzymes, such as chymase, tonin, and D and G cathepsin. Chymase, which is considered to be the most important enzyme, is a serine protease stored in the secretory granules of mast cells, which under normal conditions are present in mucosal, epithelial, and vascularized tissues, but not in circulating blood (Krystel-Whittemore et al. 2016).

The endogenous heptapeptide angiotensin (Ang)-(1–7), derived from Ang-II through the influence of angiotensin converting enzyme 2 (ACE2), modulates baroreflex sensitivity (Wessel et al. 2007). Opposite to Ang-II, Ang-1–7 couples to the G-protein MAS receptor (Santos et al. 2003) and exerts protective effects (Ferreira et al. 2012), i.e., lessened inflammation of atherosclerotic plaques (Fraga-Silva et al. 2014), vasodilatation via activation of NO, and decreased fibrosis perhaps through activation of NO and suppression of ROS generated by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (Gwathmey et al. 2010; Sampaio et al. 2007). The Ang-1–7 receptor agonist AVE0991 has been reported to alter the circadian rhythm of baroreflex in spontaneously hypertensive rats, implying the involvement of Ang-1–7 in the regulation of cardiovascular circadian rhythms (Wessel et al. 2007). Because ACE2 is a receptor for certain coronavirus strains, including the deadly one that causes coronavirus disease 2019 (COVID-19), the role of Ang-1–7 is an interesting molecule potentially useful for treating COVID-19 and its cardiovascular complications (Sahu et al., 2022).

The release into circulation of the hormone aldosterone from the adrenal cortex enhances reabsorption of sodium and water by kidney tubules (Rautureau et al. 2011). Additionally, aldosterone participates in processes that promote vascular inflammation, oxidative stress, fibrosis, endothelial dysfunction, and structural remodeling, particularly in the presence of elevated plasma sodium chloride concentration (Park and Schiffrin 2002). In endothelial cells and VSMCs, aldosterone, with influence of Ang-II, exerts effects via mitogen-activated protein kinase (also termed extracellular signal regulated kinase) and cellular Src kinase (a non-receptor tyrosine kinase), plus it partakes in epidermal growth factor receptor transactivation (Mazak et al. 2004; Min et al. 2007; Nakano et al. 2005). Aldosterone thus increases oxidative stress in VSMCs and negatively impacts endothelial function, most likely through reducing the bioavailability of NO (Nakano et al. 2005), and it induces vascular inflammation in endothelial cells through enhancing expression of intercellular adhesion molecule 1 (ICAM-1) and leukocyte adhesion (Caprio et al. 2008).

Ang-II, itself, induces endothelial dysfunction and inflammation, two fundamental processes of atherogenesis. It provokes oxidative stress through induction of ROS, like superoxide anions (O2−), which is catalyzed by NADPH oxidase, to elevate endothelial cell Ca2+ concentration and stimulate calmodulin via interaction with the Nox5/Ca2+ calmodulin binding domain (Montezano et al. 2010; Piqueras and Sanz 2020). Nox5, a novel NADPH oxidase that generates superoxide, is highly expressed in coronary vessels of CAD patients (Guzik et al. 2008; Gray and Jandeleit-Dahm 2015). Oxidative and inflammatory atherogenic processes are mediated by the stimulated expression and activation of Nox5 in arterial cells and macrophages (Touyz et al. 2003). In cultured human umbilical arterial endothelial cells, Ang-II promotes activation of Nox5 to generate O2− and RhoA, and it enhances Rho-associated kinase (Escudero et al. 2015). The RhoA/ROCK pathway functions as an upstream regulator of mitogen-activated protein kinases (MAPKs – p38MAPK and ERK1/2) that promotes transactivation of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) (Piqueras and Sanz 2020), which regulates expression of pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), IL-6, chemokines, monocyte chemoattractant protein 1 (MCP-1), adhesion molecules (P-selectin, ICAM-1, and vascular cell adhesion molecule 1 [VCAM-1]), cyclooxygenase type 2 (COX-2), and angiotensinogen (Durante et al. 2012; Liang et al. 2015). NF-κB is a signal transducer for upregulation of oxidized low-density lipoprotein (oxLDL) mediated by the expression of AT1Rs through Ang-II binding (Li et al. 2000). TNF-α, released upon binding of Ang-II to AT1Rs, in combination with IL-4 can act as a paracrine molecule, causing selective adhesion of mononuclear cells to the tunica intima of arteries through increased expression of cell adhesion molecules and release of chemokines that attract mononuclear cells (Piqueras and Sanz 2020). Ang-II additionally induces expression of osteopontin, a protein of multiple functions present in macrophages, VSMCs, and epithelial cells. Osteopontin, a constituent of atheroma in association with macrophages and foam cells, recruits inflammatory and foam cells through binding to integrins (Giachelli and Steitz 2000). Ang-II up-regulates the LOX-1 gene. LOX, a transmembrane glycoprotein, is a receptor for oxLDL binding to LOX-1 in the endothelium that enhances leukocyte adhesion molecules, activates apoptosis pathways, and elevates ROS, and, as a consequence, induces endothelial dysfunction (Lubrano and Balzan 2016). In a pro-inflammatory environment, LOX-1 is positively regulated in macrophages and is associated with >40% of oxLDL uptake, thereby significantly contributing to the formation of foam cells (Kattoor et al. 2019). oxLDL also promotes ACE generation that augments Ang-II formation and LOX-1 expression, which positively regulates expression of AT1Rs as a self-perpetuating pro-atherogenic cycle. ACE inhibitor (ACEI) and AT1R blocker (ARB) medications decrease LOX-1 expression (Lubrano and Balzan 2016) to exert beneficial anti-atherogenic effects (Tousoulis et al. 2015).

RAAS circadian rhythmicity.

The major elements of the RAAS, i.e., plasma prorenin, plasma renin, plasma ACE, Ang-I, Ang-II, and aldosterone, evidence prominent circadian rhythmicity in their activity and/or concentration (Charloux et al. 1999; Cugini et al. 1981a, 1981b, 1988; Hurwitz et al. 1985; Kawasaki et al. 1990; Kool et al. 1994; Naito et al. 2009; Portaluppi et al. 1990; Tsujino et al. 2006; Veglio et al. 1987). In humans, the RAAS commences activation during the terminal hours of the sleep span and attains maximum expression of its different constituents during the initial segment of the wake span. Thus, atherogenesis through initiation and upregulation of many, if not all, of the RAAS-promoting activities mediated by Ang-II through its binding to AT1Rs are likely to be circadian rhythmic. The ACEI enalapril remediates oxidative vascular injury, attenuates NADPH oxidase activity, decreases inflammatory mediators, and upregulates antioxidant defense mechanisms in animals (Husain et al. 2015; Suarez-Martinez et al. 2014). The ACEI perindopril reduces IL-1β level in stable CAD and essential hypertension patients (Krysiak and Okopién 2012; Madej et al. 2009), and the ARB medication olmesartan significantly reduces vascular inflammation in hypertensive patients, as revealed by significantly reduced serum levels of inflammatory markers, i.e., CRP, TNF-α, IL-6, and MCP-1 (Durante et al. 2012; Fliser et al. 2004). Prolonged treatment of atherosclerotic patients with the ARB medication valsartan ameliorates carotid artery wall hypertrophy and attenuates markers of oxidative stress, inflammation, and abnormal peripheral smooth muscle function (Ramadan et al. 2016). Other clinical studies substantiate ACEI and ARB medications significantly regresses pathologic cardiac remodeling (Dell’Italia 2011; Ferrario 2016; Malmqvist et al. 2001). It is noteworthy at-bedtime/evening, in comparison to upon-awakening/morning, scheduling of valsartan, olmesartan, or candesartan so as to optimize the PK features of the medications relative to the circadian staging of the RAAS rhythm significantly impacts urinary albumin excretion (UAE), a measure of renal vascular pathology of hypertensive patients (Eguchi et al. 2012; Hermida et al. 2005a; Kario et al. 2010). Such administration-time, i.e., circadian rhythm-dependent, differences in ARB and ACEI medications are later discussed in more detail. In this regard, the primary benefits of angiotensin-converting enzyme inhibition on cardiac remodeling during sleep time have been documented in the murine pressure overload hypertrophy model by Martino et al., 2011).

DEFINTION OF HYPERTENSION IN THE CONTEXT OF ATHEROGENESIS, CHRONOTHERAPEUTIC AND CHRONOPREVENTIVE STRATEGIES

Arterial hypertension is an important predisposing condition and risk factor for the development and progression of atherosclerosis, and conversely atherosclerosis is a risk factor for hypertension (Dzau 2001). Differentiation of normotension from hypertension depends on the technology utilized to measure BP and the available evidence (vascular and non-fatal and fatal CVD outcomes) that informs SBP and DBP threshold values of medical guidelines utilized for diagnosis. In 1997, Moser (Moser 1997), based upon a critical review of the recommended diagnostic thresholds and treatment strategies of the past published hypertension guidelines, wrote, “There are few stories in the history of medicine that are filled with more errors or misconceptions than the story of hypertension and its treatment.” Recent patient outcomes research entailing around-the-clock ABPM, as opposed to single time-of-day wakening office BP measurement (OBPM), methods has provided comprehensive perspective of the 24 h variability in SBP and DBP as it relates to risk for deleterious CVD outcomes. This has led to a novel ABPM-based definition of true arterial hypertension according to the extent to which the 24 h BP pattern is altered or disturbed, i.e., extent to which the sleep-time SBP mean is elevated and sleep-time SBP relative decline is attenuated, that not only gives new insight into circadian-rhythm-based mechanisms of atherosclerosis and its harmful outcomes but opportunities for their chronoprevention, i.e., timing medical interventions according to biological rhythms to avert pathology and/or acute non-fatal and fatal events (Hermida et al. 2020d) .

The MAPEC Study (Monitorización Ambulatoria para Predicción de Eventos Cardiovasculares, i.e., ambulatory monitoring for prediction of cardiovascular events; n=3344 participants) and the Hygia Project (n>19000 participants) were designed to assess the differential clinical importance of around-the-clock ABPM versus traditional wake-time OBPM in predicting risk for future non-fatal and fatal CVD outcomes and also relevance of the timing, i.e., upon arising from sleep vs. before bedtime, of prescription hypertension medications of diverse classes in controlling SBP and DBP and preventing vascular pathology and harmful CVD outcomes (Hermida 2007, 2016; Hermida et al. 2010, 2020a). In these primary care patient-based outcomes trials, baseline OBPM and 48h ABPM (rather than 24 h ABPM to improve reliability and reproducibility of derived ambulatory BP [ABP] parameters: Hermida et al. 2013a) were conducted on patients upon recruitment to make the differential diagnosis of normotension vs. hypertension and determine the wake-time and sleep-time SBP and DBP and sleep-time SBP decline, i.e., SBP dipping (Hermida 2007, 2016). CVD event-free survival increased with the progressive reduction in the asleep SBP mean during follow-up, and this was significant for subjects with either normal or elevated BP at baseline (Hermida et al. 2011, 2013b). As in the MAPEC Study, the progressive attenuation of the asleep SBP mean during follow-up in the Hygia Project was the most significant marker of event-free survival, independent of changes during follow-up in the wake-time office or ABPM-derived mean awake SBP mean (Hermida et al. 2018a, 2018b). Additionally, the extent of the sleep-time SBP mean decline relative to wake-time SBP mean was also significantly associated with event free survival. Thus, both in the MAPEC Study and Hygia Project, the best Cox regression model found the asleep SBP mean and sleep-time relative SBP decline, in combination, to be the most significant and strongest prognostic markers of CVD morbidity and mortality (Hermida et al. 2011, 2018b). It is noteworthy that the relationship between sleep-time relative BP decline and risk for CVD events has been reported by other investigators both for hypertensive (Dolan et al. 2005; Ingelsson et al. 2006; Ohkubo et al. 2002; Salles et al. 2016; Verdecchia et al. 2011) and normotensive (Hermida et al. 2013c) persons. The astonishing reduction in CVD outcomes in the HYGIA investigation was achieved by the at-bedtime, relative to the upon-wakening, hypertension treatment strategy without compromised adherence to therapy nor increased risk of either sleep-time or wake-time hypotension, perhaps to some extent due to the recommended clinical protocol entailing repeat of 48 h ABPM eight to twelve weeks following alteration either of the dose or/and number of prescribed medications to assess success in attaining BP control goals (Hermida et al. 2020a).

A large number of published reports support improved control of BP by evening or before bedtime than upon awakening or morning dosing of hypertension therapy. Hermida et al. (Hermida et al. 2021a, 2021b) retrieved 155 trials published between 1976 and 2020 entailing data collected from a total of 23,869 hypertensive individuals treated with 37 different single and 14 dual-fixed combination BP-lowering therapies. The vast (83.7%) majority of the trials, representing 89.5% of the total number of 23,869 hypertensive participants, documented with high consistency statistically and clinically significant enhanced BP-lowering efficacy, mainly during sleep, plus other favorable effects when the hypertension medications of the different classes and their combinations were ingested at-bedtime/evening rather than upon-waking/morning. The major benefits of the at-bedtime/evening in comparison to the upon awakening/morning treatment strategy revealed by the 62 ABPM-based trials include: (i) significantly enhanced reduction of the asleep SBP mean by an average 5.12 mm Hg (P<0.001) without diminished efficacy in reducing the awake SBP mean, with the beneficial effect on sleep-time SBP regulation being markedly greater among individuals at high CVD risk, such as those requiring multiple medications to achieve adequate ambulatory BP (ABP) control and those having a medical history of resistant hypertension, type 2 diabetes, CKD, and previous medical history of a CVD event; (ii) significantly greater increased sleep-time relative SBP decline by an average of 3.23% (P<0.001) towards the normal and lower CVD risk dipper 24 h BP phenotype, with the effect being greater with fixed dual-medication combinations (5.50% increased sleep-time relative SBP decline, P<0.001) and in high CVD risk cohorts (5.29% increased sleep-time relative SBP decline, P<0.001); (iii) improved renal function – greater attenuation of urinary albumin excretion and greater increase of glomerular filtration rate (GFR) – and superior reduction of cardiac and vascular remodeling and pathology, i.e., greater regression of left ventricular mass index, left ventricular posterior diameter and relative wall thickness, and carotid artery plaque size; (iv) Similar incidence of adverse effects, or even lower incidence of adverse effects mainly when ingesting an ACEI or CCB alone or in combination with other medications; and (v) absence of sleep-time hypotension among the at-bedtime treated individuals. We believe the inability to detect treatment-time difference in effects by the small proportion (16.3%) of the 155 published trials is likely explained by deficiencies in study design and/or conduct (Hermida et al. 2021c). Perhaps, one of the most impressive and supportive finding of this systematic and comprehensive review by Hermida et al. (Hermida et al. 2021a, 2021b) is not a single published treatment-time trial, out of the 155 in total, conducted during the past 45 years reported significantly better benefit of the most conventional and most popularly advocated (Kreutz et al. 2020; Turgeon et al. 2021) upon-waking/morning hypertension therapy routine.

An extensive body of literature (for reviews see: Baraldo 2008; Bélanger et al. 1997; Bruguerolle 1988; Labrecque et al. 2003; Reinberg 1983) substantiates both the pharmacokinetics (PK) and pharmacodynamics (PD) of medications, whether their intrinsic half-life is very short or very long (≥24 h), can be influenced not only by various exogenous 24 h cycles, particularly those of posture and nutrient and liquid intake, but the circadian stage of the many involved cell, tissue, and organ rhythms. Thus, the PK of medications, including ones prescribed to manage hypertension and atherosclerosis, can be substantially affected by circadian rhythms of: (i) gastric pH, gastric emptying rate, gastrointestinal tract motility, gastrointestinal tract blood perfusion, and drug transport phenomena that influence drug absorption; (ii) drug-free fraction that depends on red and white blood cell number and plasma binding protein concentrations, cell membrane transport mechanisms, and tissue perfusion that influence drug distribution; (iii) activity of multiple enzymes, biochemical pathways of liver and other tissues and hepatic and renal blood perfusion that influence drug metabolism; and (iv) GFR and other phenomena of renal tubules and liver function and bile that influence drug elimination. Thus, even the constant infusion of medications of different classes can exhibit meaningful predictable-in-time 24 h variation in their PK (e.g., Decousus et al. 1985a, 1985b; Petit et al. 1988; Queneau et al. 1985; Smolensky et al. 1984; White et al. 1991). Nonetheless, dosing-time disparities in the PD of medications need not be dependent solely on circadian rhythms of processes deterministic of PK; they can result independent of or in addition to dosing-time differences in the PK as a consequence of circadian processes in medication-targeted cells, tissues, and organs. This is demonstrated by clinically significant differences in the response of stable asthmatic subjects to the direct targeting of the airways by inhalation at different times during the 24 h of aerosolized agents, like acetylcholine, adrenaline, histamine, isoproterenol, and methacholine (Barnes et al. 1982; Brown et al. 1988; DeVries et al. 1962; Jarjour 1999; Martin et al. 1990; Reinberg et al. 1971) and by temporal differences in the blood concentration-effect relationship of a wide variety of medications when infused at a constant rate for ≥24 h, e.g., low molecular weight heparin, H₁-receptor blocker ranitidine, adenosine receptor antagonist aminophylline, among other medications (Decousus et al. 1985a, 1985b; Reinberg 1983; Smolensky et al. 1986; White et. al. 1991). Indeed, numerous trials demonstrate rather profound ingestion-time disparities in BP-lowering by medications of the different classes of hypertension therapy (Hermida et al. 2020c, 2020b, 2021a, 2021b; Smolensky et al. 2010, 2015).

Earlier in this article, we presented evidence that these classes of therapies administered to laboratory animals and humans, even without regard to circadian time, suppress atherogenesis and ameliorate atherosclerotic vascular pathology mediated through Ang-II/AT1Rs coupling and aldosterone, primary elements of the RAAS. Specifically, they induce: remediation of markers of oxidative stress and oxidative vascular injury, attenuation of NADPH oxidase activity, diminution of inflammatory mediators and vascular inflammation, upregulation of antioxidant defense mechanisms, improvement of peripheral smooth muscle function, lessening or regression of pathologic cardiac remodeling, and regression of carotid artery wall hypertrophy (Dell’Italia 2011; Durante et al. 2012; Ferrario 2016; Fliser et al. 2004; Husain et al. 2015; Krysiak and Okopién 2012; Madej et al. 2009; Malmqvist et al. 2001; Ramadan et al. 2016; Suarez-Martinez et al. 2014).

One important, but seemingly under-appreciated finding of the Hygia Project was the profoundly better protection against non-fatal and fatal CVD outcomes achieved by the long-acting ARB and ACEI classes of medications in comparison to other popular classes of long-acting hypertension medications, i.e., ß-blockers, diuretics, and CCBs, when routinely ingested at-bedtime rather than upon-awakening (Hermida et al. 2020b). The results of the MAPEC Study and Hygia Project both showed through the application of 48 h ABPM that the asleep SBP mean and extent of SBP dipping are the strongest predictors of vascular morbidity and mortality. This has led us to conclude that around-the-clock ABPM, preferably of 48 h duration (Hermida et al. 2013a) should be the basis for making the differential diagnosis of normotension versus hypertension, and, furthermore, that these two ABP parameters in combination should be considered as the true definition of arterial hypertension (Hermida et al. 2018a, 2020d).

SLEEP-WAKE CIRCADIAN RHYTHM DISORDERS AND ATHEROSCLEROSIS

Disordered sleep can contribute to the development and progression of atherosclerosis and CVD-associated vascular pathology with elevated CVD risk (Figure 3) through increasing the sleep-time SBP and lessening the sleep-time SBP relative decline, manifesting as non-dipper and riser BP 24 h phenotypes (Cuspidi et al. 2019; Kario 2009; Loredo et al. 2001; Ma et al. 2017; Sekizuka et al. 2018). The normal sleep-wake state alternation-associated BP 24 h variation derives from multiple behavioral and environmental cycles of noise, light, posture, activity, and nutrient and stimulant intake, plus numerous endogenous circadian rhythms (Smolensky et al. 2017). The mechanisms underlying the core body temperature (CBT) circadian rhythm serve as important regulators of the sleep/wake 24 h periodicity, and they also contribute to the BP circadian rhythm (Haghayegh et al. 2021). Some four decades ago, Borbély introduced a model of sleep regulation comprised of two interacting processes, homeostatic Process S and circadian Process C (Borbély 1982, Borbély and Achermann 1999; Borbély et al. 2016). Process S, indicative of sleep pressure, progressively accumulates during wakefulness and progressively dissipates during sleep. Electroencephalography-assessed theta activity during waking is a biomarker of the rising limb of Process S, and non‐rapid eye movement slow wave sleep is a biomarker of its descending limb (Borbély and Achermann 1999), although blood levels of hypoxanthine and thromboxane A2 have also been suggested as biomarkers of Process S (Sher et al. 2021). Process C derives principally from the central circadian pacemaker, the suprachiasmatic nuclei (SCN), mediated primarily through the CBT 24 h rhythm. The CBT rhythm, which is additionally controlled by peripheral clocks that modulate thermogenic metabolic processes, is characterized by major nadir during deepest sleep, small rise just before sleep offset (spontaneous wakening), greater gradual rise during wakefulness -- although with a small mid-wake span dip, absolute peak approximately 4–6 h before the conclusion of wakefulness, and discernable decline just prior to sleep onset (Aschoff 1983). The decline at the end of the activity span in CBT when Process S is at threshold is a necessary biological prerequisite for sleep induction. There is strong relationship between the circadian rhythms of thermoregulatory CBT Process C and arterial BP. The marked CBT decline just before and during sleep results from the redistribution of 25–50% of the cardiac output from the systemic circulation to the arteriovenous anastomoses (AVAs) of the glabrous skin, most notably the skin of the palms of hands and soles of feet, to facilitate dissipation of body heat to the external environment (Hayhayegh et al. 2021; Taylor et al. 2014). This substantial redistribution of the cardiac output in normal sleepers along with various circadian rhythms (Smolensky et al. 2017) mediate the normal decline in SBP and DBP and normal dipper SBP 24 h phenotype.

Figure 3. Disturbance of circadian rhythms, clock gene expression, deregulation of RAAS/AR and risk for cardiovascular health and disease.

RAAS, renin-angiotensin-aldosterone system; AR, adenosine receptor.

The sleep of humans undergoes characteristic age-related changes, including increased sleep fragmentation and fragility, reduced stage N3 slow wave sleep (SWS), and increased lighter NREM stages N1 and N2 sleep (Mander et al. 2017). Old, as compared to young, adults also show shorter and fewer NREM-REM sleep cycles, more time spent awake after sleep onset, and circadian phase advancement. Sleep maintenance insomnia and delayed sleep onset (Lack et al. 2008; Raymann et al. 2005), OSA (Martinez-Nicolas et al. 2017), restless leg syndrome/periodic limb movements in sleep (Maiolino et al. 2021), and vasospastic syndrome (Pache et al. 2001; Vollenweider et al. 2007) disorders tend to be associated with altered Process C, i.e., disturbed CBT circadian rhythmicity, manifested either as an altered staging and/or attenuated before-sleep decline of CBT. Such abnormalities of the CBT circadian rhythm are likely the result, at least to some extent, of lower than normal redistribution of the cardiac output to the AVAs of the glabrous skin for body heat dissipation, which may contribute to elevated asleep SBP and non-dipper or even riser circadian SBP hypertensive phenotypes prevalent in these and perhaps other sleep disorders.

Sleep duration, shift work, and risk for atherosclerosis and metabolic syndromes

Shift work may promote atherosclerosis and increase the risk for coronary heart disease. A human study involving 65 shift workers and 29 day workers has demonstrated that compared to day workers, shift workers had increased thickness of carotid arterial wall and CRP levels (Skogstad et al. 2019), which are indicative of plaque formation and inflammation, respectively. Furthermore, Figueiro, et al. (2021) examined the light-dark patterns resembling those experienced by humans working day shifts or rotating shift schedules, in low-density lipoprotein receptor (LDLR) knockout mice, a widely used murine model of hypercholesterolemia and atherosclerosis. The animals under day shifts were maintained under a regular 12 h of light and 12 h of dark cycle. The investigators showed experimentally that the shift work paradigm disrupted the foam cell's molecular clock and increased endoplasmic reticulum stress, and apoptosis, and it significantly increased plaque necrotic core areas.

There are disparities between investigations regarding the impacts of sleep duration and alignment on markers of atherogenesis and CVD risk (Butler et al. 2020; Cherubini et al. 2021; Gupta et al. 2021; Li et al. 2020, 2021). Although studies substantiate relationship between sleep duration and CVD risk (e.g., Covassin and Singh 2016; Kwok et al. 2018; Tao et al. 2021), ones linking it specifically to subclinical atherosclerosis are relatively sparse. Domínguez et al. (2019) conducted wrist actigraphy on 3,974 middle-age male participants to categorize sleep duration, i.e., very short sleep duration of <6 h, short sleep duration of 6 to 7 h, reference sleep duration of 7 to 8 h, and long sleep duration of >8 h. Adjusting for conventional risk factors, very short sleepers relative to the normal reference sleepers showed higher atherosclerotic burden (odds ratio: 1.27; 95% confidence interval: 1.06 to 1.52; P = 0.008) in terms of non-coronary atherosclerosis and coronary calcification assessed by carotid and femoral 3-dimensional vascular ultrasound and cardiac computed tomography. Kundel et al. (2021) utilized data of the Multi-Ethnic Study of Atherosclerosis-SLEEP and Multi-Ethnic Study of Atherosclerosis-PET ancillary investigation to assess association between sleep duration and carotid inflammation, defined by target-to-background ratios, and measures of carotid wall remodeling, i.e., carotid wall thickness. Average sleep length of short duration sleepers was 5.1 ± 0.9 h/night, with 31% of the entire study sample sleeping <6 h/night, and it was 7.1 ± 0.8 h/night in the normal duration sleepers, the remaining 69% of the study sample. Prevalence of pathologic vascular inflammation, determined as the maximal target-to-background ratio >1.6, was greater in the short than normal duration sleepers (89% vs. 53%; P = .01). Blasco-Colmenares et al. (2018) explored the association between sleep duration, determined by questionnaire, and coronary artery calcium score (CACS), assessed by computed tomography, and presence of carotid plaque and femoral plaque, assessed by ultrasound, of 1968 middle-age men participating in the Aragon Workers' Health Study (AWHS). Odds ratios (95% CI) for CACS >0 for sleep durations of ≤5, 6, and ≥8 h, with 7 h as reference, were 1.34 (0.98–1.85), 1.35 (1.08–1.69,) and 1.21 (0.90–1.62), respectively (P = 0.04). CACS ≥100 and CACS across the three sleep duration groups displayed a similar U-shaped relationship. However, non-statistically significant differences were found in the odds ratios for presence of ≥1 carotid plaque for sleep duration of ≤5, 6, and ≥8 h, with 7 h as reference, i.e., 1.23 (0.88–1.72), 1.09 (0.86–1.38), and 0.86 (0.63–1.17), respectively (p = 0.31), and presence of ≥1 femoral plaque, i.e., 1.25 (0.87–1.80), 1.19 (0.93–1.51), and 1.17 (0.86–1.61), respectively (P = 0.39). Finally, Hijmans et al. (2019) investigated whether levels of circulating microRNAs (miRNAs) identified as biomarkers of vascular inflammation and endothelia dysfunction are altered in adult short ,<7 h/night, sleepers. A total of 24 persons were studied, 12 with normal nightly sleep duration of ≥7.0 h/night and 12 with short nightly sleep duration of <7 h/night. Circulating levels of miR-125a (3.07 ± 1.98 vs. 7.34 ± 5.34 a.u.), miR-126 [1.28 (0.42–2.51) vs. 1.78 (1.29–4.80) a.u.], and miR-146a [2.55 (1.00–4.80) vs. 6.46 (1.50–11.44) a.u.] were significantly lower, respectively by ~60, 40, and 60%, in short than normal sleepers, suggesting involvement of these miRNAs in vascular inflammation and endothelial dysfunction. Bain et al. (2017) additionally found chronic short sleep duration in middle-aged adults to be associated with a measure of endothelial dysfunction, NO-mediated endothelium-dependent vasodilation. Forearm blood flow response to acetylcholine was lower by ~20% (P<0.05) in individuals who habitually slept on average ~6 h/night (from 4.6 ± 0.3 to 11.7 ± 1.0 ml/100 ml tissue/min) than those who on average habitually slept ~7.7 h/night (from 4.4 ± 0.3 to 14.5 ± 0.5 ml/100 ml tissue/min). Furthermore, the endothelial NO synthase inhibitor NG-monomethyl-L-arginine as expected significantly reduced the FBF response of normal sleepers to acetylcholine by ~40%, but not of short sleepers.

Several human investigations (Cherubini et al. 2021; Dutheil et al. 2020; Jankowiak et al. 2016; Kang et al. 2016; Peñalvo et al. 2021; Rizza et al. 2020; Skogstad et al. 2019; Sugiura et al. 2020) involving rotating and night shift workers have demonstrated that chronic circadian misalignment of the sleep-wake 24 h cycle is associated with subclinical vascular pathology of atherosclerosis. However, because of several uncontrollable complicating factors, such as diet, smoking, alcohol consumption, sleep timing and duration, and comorbidities, it is difficult to discern the relative contribution of these and other external factors versus circadian misalignment, per se, to the signs of subclinical atherosclerosis. Therefore, laboratory-based animal and human simulated shiftwork studies are necessary. Using calibrated devices, Figueiro et al. (2021) exposed LDLR-knockout mice to a stimulated, rotating shift work schedule. Their data show that the shiftwork LDLR-knockouts have a disrupted molecular clock of lipid-laden foam cells as well as increases in apoptosis and stress of the endoplasmic reticulum. Moreover, plaque lesions of the shift work mice were larger and contained less pro-stabilizing fibrillar collagen and significantly increased necrosis. Another laboratory animal study by Schilperoort et al. (2020) exposed hyperlipidemic APOE*3-Leiden.CETP mice either to regular light-dark cycles, weekly 6 h phase advances or delays, or weekly alternating 12 h shifted light-dark cycles to simulate shift work schedules. Animals exposed for 15 weeks of alternating shift-work-like light-dark cycles experienced severe circadian disruption and displayed marked increase in atherosclerosis, with ~2-fold increase in plaque lesion size and severity. In contrast, animals that experienced phase advances and delays showed only mild circadian disruption and displayed no significant effect on atherosclerosis development. Additionally, the investigators identified increased markers for inflammation, oxidative stress, and chemoattraction in the vessel wall of the animals exposed to the stimulated shift work schedule. Morris et al (2016, 2017) conducted a series of laboratory simulated shiftwork investigations involving both non-shift-workers and actual shift workers. In one of their studies non-shift worker participants were subjected to two 8-day laboratory protocols entailing 12 h inverted behavioral and environmental cycles for 3 days to investigate effects upon subclinical biomarkers of atherosclerosis. The induced circadian misalignment was associated with increased SBP and DBP 24 means by 3.0 mmHg and 1.5 mmHg, resulting primarily from increased BP during sleep opportunities, i.e., SBP by 5.6 mmHg and DBP by 1.9 mmHg, and to lesser extent by increased BP during wake periods, i.e., SBP by 1.6 mmHg and DBP by 1.4 mmHg. Circadian misalignment additionally increased 24 h serum interleukin-6, C-reactive protein, resistin, and tumor necrosis factor-α levels by 3–29%.

The meta-analysis of 34 studies representing in total 2,011,935 people by Vyas in 2012 (BMJ) has revealed significantly increased risk of ischemic heart disease and stroke risk in shift workers. Shift work appears to be associated with increased coronary events (risk ratio 1.24, 1.10 to 1.39), myocardial infarction (risk ratio 1.23, 95% confidence interval 1.15 to 1.31; I(2)=0), and ischaemic stroke (1.05, 1.01 to 1.09; I(2)=0), albeit with significant heterogeneity across studies (I(2)=85%). In this regard, a case control human study conducted by Skogstad et al. (2019) showed evidence of increased thickness of carotid arterial wall and C-reactive protein (CRP) levels in shift workers (Skogstad et al. 2019). Very recently, Mason et al. (Mason et al. 2022) investigated whether acute exposure to light during nighttime sleep adversely affects next-morning glucose homeostasis in 20 young adult participants; 10 of them were exposed to one night of sleep in dim light (<3 lx) followed by one night of sleep with overhead room lighting (100 lx), i.e., room-light condition, and the other 10 were exposed to two consecutive nights of sleep in dim light, i.e., dim-light condition. Measures of insulin resistance (morning homeostatic model assessment of insulin resistance, 30 min insulin area under the curve [AUC] from a 2 h oral glucose tolerance test) were higher in the room light vs. dim light condition, while melatonin levels were similar in both conditions. In the room light condition, participants spent proportionately more time in stage N2 and less in slow wave and rapid eye movement sleep. Heart rate was higher and heart rate variability lower (higher sympathovagal balance) during sleep in the room-light vs. the dim-light condition. Importantly, the higher sympathovagal balance during sleep was associated with a higher 30 min insulin AUC, consistent with increased insulin resistance the following morning. Thus, the data suggest that exposure to room light during sleep may impair glucose homeostasis, potentially via increased SNS activation, and thereby foster processes of artherosclerosis.

The collective findings of animal and human studies imply that circadian misalignment caused by rotating shift work may trigger hypertension, metabolic disorder, and atherosclerosis as well as increase the risk for coronary heart disease and stroke.

Obstructive sleep apnea (OSA) and risk for cardiovascular disease

OSA is associated with risk for arterial hypertension (Cuspidi et al. 2019; Durán-Cantolla et al. 2010; Kario 2009; Loredo et al. 2001; Ma et al. 2017; Martínez-García et al. 2013; Muxfeldt et al. 2015; Peppard et al. 2000; Sekizuka et al. 2018); additionally, it is a risk factor for atherosclerotic vascular disease risk (Deol et al. 2020; Gunnarsson et al. 2014; Kim et al. 2020; Souza et al. 2021; Toraldo et al. 2013). OSA is a chronic sleep disorder characterized by repeated episodes of airway obstruction with increased ventilatory effort, decreased oxygen saturation, and frequent cortical arousals, and it is often associated with resistant hypertension (i.e., SBP and/or DBP higher than medical guideline thresholds when treated with three prescription BP-lowering medications with one bring a diuretic). OSA-mediated pathophysiology is mediated by repetitive hypoxic insults and exaggerated fluctuations in intra-thoracic pressure that negatively impact cardiovascular hemodynamics and trigger atherogenic endothelial dysfunction, vascular inflammation, and sympathetic activation. The Wisconsin Sleep Cohort 13-year follow-up study clearly demonstrated OSA is associated with subclinical carotid artery disease (Gunnarsson et al. 2014). In this prospective population-based cohort study, 790 randomly selected employed Wisconsin residents completed a mean of 3.5 (range 1–6) polysomnograms. OSA severity was characterized by the AHI (number of apnea and hypopnea events per hour of sleep). Common carotid artery intima thickness (IMT) and plaque presence, both sensitive markers of atherosclerosis, were assessed by B-mode ultrasound. Adjusting for age, sex, body-mass index, SBP, smoking, and use of lipid-lowering, antihypertensive, and diabetes medications, baseline AHI independently predicted future carotid IMT, plaque presence, and plaque score (odds ratio 1.30 [1.05–1.61]; P=0.018). In cumulative risk factor-adjusted models, AHI independently predicted future carotid plaque presence (P=0.012) and score (P=0.039). In the ELSA-Brazil Study (Souza et al. 2021), a large cohort of participants (n=2009) underwent clinical evaluation, sleep study, and one-week of wrist actigraphy to elucidate the independent associations between OSA and sleep duration as well as to assess potential inflammatory and metabolic mediators of carotid intima-media thickness (CIMT). Compared to participants without OSA (AHI <5 events/h; n=613), those with mild (AHI 5–14.9 events/h; n=741), moderate (AHI 15–29.9 events/h; n=389), and severe OSA (AHI ≥30 events/h; n=266) evidenced progressive increase in CIMT (respectively, 0.690 [0.610–0.790] mm, 0.760 [0.650–0.890] mm, 0.810 [0.700–0.940] mm, and 0.820 [0.720–0.958] mm; P<0.001). In contrast, CIMT was found to be independent of sleep duration, being similar for those who slept <6 h (0.760 [0.650–0.888] mm), 6 to 8 h (0.750 [0.640–0.880] mm), and >8 h (0.740 [0.670–0.900] mm). All forms of OSA were independently associated with CIMT (mild: β=0.019, SE 0.008; P=0.022; moderate: β=0.025, SE 0.011; P=0.022; severe: β=0.040, SE 0.013; P=0.002). Moreover, the association between AHI with CIMT was mediated by increased blood concentration of CRP, a marker of inflammation, and also triglycerides, a measure of dyslipidemia (P<0.01). Sleep duration did not interact with severity of OSA and CIMT. Thus, OSA was found to be independently associated with increased CIMT in a dose-response relationship partially mediated by inflammation and dyslipidemia. In contrast, sleep duration was found neither to be associated nor an interacting factor with OSA that increases CIMT. Although the ELSA-Brazil Study (Souza et al. 2021) did not find sleep duration to be associated as an interacting factor of CIMT, the study by Lao et al. found sleep duration and other characteristics of sleep to be associated with coronary heart disease (Lao et al. 2018). Participants of a Taiwanese cohort of 60,586 participants ≥40 years of age completed a questionnaire to collect information on coronary heart disease events, sleep quality, sleep duration, and potential confounders. A total of 2,740 participants reported new coronary heart disease events at follow-up. Cox regression analysis revealed a statistically significantly HR for coronary heart disease events in those having a sleep duration of <6 h/d (HR: 1.13 [1.04–1.23]), but not in those having a sleep duration of >8 h/d (HR: 1.11 [0.98–1.26]). Dreamy sleep (HR: 1.21 [1.10–1.32]) and difficulty falling asleep/use of sleep medications/drugs also associated with increased risk of coronary heart disease (HR: 1.40 [1.25–1.56]).

Continuous positive airway pressure (CPAP) is commonly prescribed to improve sleep quality and associated elevated BP of OSA. Becker et al. reported effective treatment of OSA with CPAP may significantly reduce the 24 h mean arterial as well as daytime and nighttime BP by as much as 10 mm Hg (Becker et al. 2003). However, most published prospective studies and meta-analyses report CPAP exerts only slight effect on BP, whether assessed by wake-time OBPM or by around-the-clock ABPM. Indeed, most CPAP trials report only a 2–4 mm Hg reduction of wake-time OBPM or ABP 24 h, “daytime”, and “nighttime” SBP or DBP means in OSA patients, which in some of them is not statistically significant different from the amount of change in BP of non-CPAP controls. Moreover, CPAP is typically associated with only slight improvement of the ‘nighttime’ BP decline, on average by ~2%, and incidence of non-dipper 24 h BP patterning (Alajmi et al. 2007; Bratton et al. 2015; Durán-Cantolla et al. 2010; Hu et al. 2015; Hui et al. 2006; Liu et al. 2016; Muxfeldt et al. 2015; Pépin et al. 2010; Schein et al. 2014). The observed relatively small BP-lowering effects of CPAP may be the consequence of poor adherence, since the amount of BP reduction has been reported to vary with duration of utilization, and optimization of CPAP settings. It is, therefore, noteworthy that clinically meaningful reduction of BP by CPAP in OSA patients in some studies has been found to be achieved only when combined with prescribed hypertension medication (Crippa et al. 2016; Hu et al. 2015; Pépin et al. 2014). In this regard, Pépin et al. found the combination of the ARB valsartan (presumably ingested in the morning) plus CPAP use during sleep by hypertensive OSA patients resulted in a four-fold greater reduction of the 24 h BP mean compared to CPAP, alone (Pépin et al. 2014). Furthermore, Crippa et al. found bedtime ingestion of 10 mg barnidipine, a dihydropyridine calcium antagonist, added to an established (presumably upon-awakening) regimen of hypertension medications completely normalized the BP circadian rhythm of 78% of OSA patients untreated by CPAP due to contraindications or rejection of its use (Crippa et al. 2016). Unfortunately, the study protocol did not include a morning treatment arm for the added 10 mg barnidipine therapy to appropriately judge the true advantage of the at-bedtime dosing scheme. Based on the previously discussed findings of both the MAPEC Study and Hygia Project, the combination of CPAP during sleep plus at-bedtime (rather than upon-awakening or morning) hypertension pharmacotherapy, preferably entailing an ARB or ACEI, is expected to substantially improve the control of the asleep SBP and DBP and their 24 h patterning toward normal, even in difficult to control resistant hypertensive OSA patients (Yap et al. 2020).

Mokros et al. measured BP on three occasions, i.e., first clinical visit to the sleep medicine clinic and evening before and morning after polysomnography, to predict both the severity of OSA in relation to DBP and CVD risk. Morning measurement of BP was predictive of OSA severity of individuals not found to be hypertensive by wake-time OBPM. Individuals with severe OSA (AHI ≥30 events/h) presented with higher morning DBP than non-OSA healthy controls (AHI <5 events/h) and those suffering from mild (15< AHI ≥5 events/h) and moderate OSA (30 < AHI ≥15 events/h): 86.2 ± 11.3 vs. 79.2 ± 8.5, 80.3 ± 10.2 and 81.4 ± 9.6 mm Hg, respectively (P<.01). In a linear regression model, morning DBP was predicted by AHI (ß = 0.14, P < .001) and body mass index (BMI) (ß = 0.22; P < .01), but not by age (ß = 0.01; P = .92), male sex (ß = -0.06; P = .19), or smoking (ß = 0.01; P = .86) (Mokros et al. 2017). The study by Mokros et al. relied only on traditional single time-of-day OBPM plus before and after polysomnography BP assessments rather than around-the-clock ABPM. The results of the MAPEC Study and Hygia Project suggest traditional cuff BP measurements are not predictive of risk for non-fatal and fatal CVD events when the asleep SBP mean and extent of SBP 24 h dipping are entered into Cox survival analyses, thereby supporting the contention the diagnosis of arterial hypertension should be based on these two ABPM-derived variables (Hermida et al. 2018a), and suggesting, furthermore, OBPM and other wake-time cuff measurements are unlikely to properly assess the BP of OSA patients as accurately as around-the-clock ABPM. Therefore, we propose ABPM be standard procedure to assess the sleep-time and wake-time BP means and 24 h BP phenotype of OSA and other sleep disordered persons (Castriotta 2017).

DISCUSSION

Predisposing, causative, and conditional risk factors of atherosclerosis include family history, age, male sex, lifestyle (sedentary habits, poor diet, tobacco use, excessive alcohol consumption, and emotional stress), obesity, metabolic dysfunction (insulin resistance/type 2 diabetes and dyslipidemia - elevated low density and diminished high density cholesterol), hypertension, and OSA. Attenuation of the risk factors of atherosclerosis can be achieved by improving environmental, behavioral, and lifestyle factors, such as by choosing a healthy diet, participating in exercise, reducing body weight, ceasing smoking, moderating alcohol intake, and mitigating stress. Many biological risk factors and mechanisms of atherosclerosis and its complications exhibit circadian rhythms that are controlled by interactions between clock gene products and environmental factors. The findings of recent chronobiology and chronotherapy studies have shed new insight into the chronopathology and management of atherosclerosis. Indeed, it has been ably demonstrated the circadian organized RAAS, primarily mediated though Ang-II and its coupling with AT1Rs and stimulation of aldosterone - activities that increase in intensity between the terminal hours of the sleep span and initial span of wakefulness - plays a major role in atherogenesis. A considerable number of studies have demonstrated that ARBs and ACEIs, even when administered without attention to circadian time, ameliorate biomarkers of atherogenesis and lessen the risk for associated pathological sequelae. Enalapril in laboratory animals remediates oxidative vascular injury, attenuates NADPH oxidase activity, decreases inflammatory mediators, and upregulates antioxidant defense mechanisms (Husain et al. 2015; Suarez-Martinez et al. 2014); perindopril in stable CAD and essential hypertension patients reduces IL-1β level (Krysiak and Okopién 2012; Madej et al. 2009); olmesartan in hypertensive patients significantly reduces vascular inflammation (Durante et al. 2012; Fliser et al. 2004); and valsartan in patients with abnormal CIMT ameliorates carotid artery wall hypertrophy and improves markers of oxidative stress, inflammation, and peripheral smooth muscle function (Ramadan et al. 2016). Other clinical studies substantiate medications of these classes also remediate the pathologic cardiac remodeling of atherosclerosis (Dell’Italia 2011; Ferrario 2016; Malmqvist et al. 2001). The proper scheduling of ARB and ACEI therapy to the circadian staging of the RASS shows promise of better slowing or averting tissue remodeling and progression of atherogenesis, with possible restoration to some extent of vascular and organ dysfunction. The results of the MAPEC Study and Hygia Project clearly elucidate for hypertensive individuals the at-bedtime, rather than upon-awakening, treatment schedule of ARB and ACEI medications better ameliorates elevated asleep SBP and non-dipper 24 h pattering, beneficial effects that have been shown to significantly reduce the risk for vascular pathology, CKD, and non-fatal and fatal CVD outcomes. Although these beneficial effects of ARBs and ACEIs are interpreted to result principally from improved BP control and 24 h patterning, we hypothesize they represent to greater extent their suppressive effects upon atherogenic mechanisms and processes mediated by elements of the RAAS. This hypothesis awaits future testing. In the United States ß-blocker and diuretic medications are popular low-cost therapies prescribed to manage hypertension and attendant CVD risks. However, if it is substantiated the more meaningful beneficial effects of antihypertension therapy derive most extensively from anti-atherogenic actions, rather than numerical reduction of SBP and DBP, then the optimal management of atherogenesis in the context of CVD risk seemingly entails in combination proper choice of class, dose (ARBs or ACEIs), and circadian timing (at-bedtime) of medications that exert strongest prevention.

Aspirin (acetylsalicylic acid, ASA), used in the primary and secondary prevention of CAD, is thus relevant to the medical management of atherosclerosis. ASA is a potent antioxidative agent that reduces vascular production of superoxide, suppresses prostaglandin synthetase activity, inhibits renin formation – the rate-limiting sleep of the classic RAAS pathway of Ang-II generation, provokes NO release, and constrains platelet aggregation and other functions (Bonten et al. 2014, 2015; Brooks et al. 1980; Buurma et al. 2019; Krasińska et al. 2019; Magagna et al. 1994; Snoep et al. 2009; Zhou et al. 2020). Evidence the biological effects of low-dose ASA are administration-time-dependent was initially reported by one of us authors (RCH) though a series of investigations involving participants diagnosed with mild hypertension (Ayala and Hermida 2010; Hermida et al. 1994, 1997, 2003, 2005b, 2005c) that consistently substantiated superior BP-lowering effect when routinely ingested at-bedtime than upon-awakening. In the largest study, 130 male and 186 female untreated mild hypertensive participants 44.1 ± 13.2 years of age were randomized to ingest aspirin (100 mg/day) either upon-awakening or at-bedtime daily for three months. BP was measured for 48 h immediately before and after the three-month treatment span. Dosing of 100 mg/daily of aspirin at-bedtime, in comparison to upon-awakening, resulted in statistically significant greater average reduction in SBP and DBP by 7.2 and 4.9 mmHg, respectively, with the reduction of the sleep-time BP mean in hypertensive non-dippers (11.0 mmHg SBP; 7.1 mmHg DBP) being twice as the amount than in dippers (5.5 mmHg SBP; 3.3 mmHg SBP; P<0.001). Interestingly, the reduction in BP was significantly better after ASA ingestion at-bedtime by women (8.0 mmHg SBP; 5.6 mmHg DBP) than men (5.5 mmHg SBP; 3.4 mmHg DBP; P<0.009 between men and women). Other investigations indicate evening, in comparison to morning, ingestion of low-dose aspirin results in enhanced desired effect upon platelets. For example, Krasińska et al. in a study of 175 CAD and arterial hypertensive patients found at-bedtime ingestion of ASA resulted in greater reduction in platelet aggregation, with the benefit gained by changing its administration from morning to evening, again being greater in women than men (Krasińska et al. 2019). Bonten et al. found at-bedtime, compared with upon-awakening, low dose 80 mg aspirin ingestion by healthy subjects better reduces COX-1-dependent platelet reactivity during the initial hours of wakefulness (Bonten et al. 2014), when non-fatal and fatal thrombotic events in at-risk persons are most frequent (Muller 1999; Muller et al. 1989). A second larger study by the same group involving 133 patients found ingestion of 100 mg aspirin at-bedtime versus upon-awakening resulted in significantly greater reduction in platelet reactivity (Bonten et al. 2015), with similar finding reported by Buurma et al. (Buurma et al. 2019). However, the meta-analyses performed by Zhou et al. (2020), while confirming the ingestion of low-dose aspirin before bedtime better reduces SBP and DBP than ingestion in the morning, did not confirm treatment-time differences in the suppression of platelet aggregation or platelet function parameters, perhaps due to the large heterogeneity across the considered studies (Zhou et al. 2020). Although the intensity of the multiple actions of low-dose aspirin are suggested to differ according to its timing, no clinical outcomes studies have yet been conducted to assess whether evening/at-bedtime than morning/upon-awakening ingestion results in better protection against the occurrence of non-fatal and fatal CVD outcomes.

Hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors, also known as statins, are frequently prescribed to treat hypercholesterolemia, an important risk factor for atherogenesis and life-threatening and life-ending CVD outcomes. Statins reduce total cholesterol, low-density lipoprotein cholesterol, and triglyceride concentrations, and they increase high-density lipoprotein cholesterol. Conversion of 3-hydroxy-3-methyl glutaryl-CoA (HMG-CoA) to mevalonate in hepatocytes is mediated by the enzyme HMG-CoA reductase, the first and rate-limiting step in cholesterol biosynthesis, which exhibits circadian rhythmicity. The therapeutic effect of statins derives from their competitive inhibition of the HMG-CoA reductase enzyme. As reviewed elsewhere by some of us authors (Smolensky et al. 2021), medications prescribed for hypercholesterolemia, such as the monotherapies of ezetimibe, fluvastatin, lovastatin, niacin, pravastatin, and simvastatin and the combination therapies of ezetimibe/simvastatin, lovastatin/niacin, and niacin/simvastatin, are recommended for ingestion toward or at the conclusion of the daily wake span, the circadian time of heightened activity of the HMG-CoA reductase enzyme, so as to exert best beneficial effect.

OSA is a risk factor for atherosclerosis and non-fatal and fatal CVD events. CPAP is considered first line treatment for OSA. However, typically it does not sufficiently attenuate the sleep-time SBP mean or normalize the non-dipper SBP pattern, especially in poorly adherent and resistant hypertensive OSA patients. This suggests the need of prescription BP-lowering therapy, preferably an ARB or ACEI at-bedtime, based upon the substantial literature reviewed herein demonstrating the enhanced anti-angiogenic effects of these classes of medications and better protection against non-fatal and fatal CVD events when ingested at this time, as demonstrated by the findings of both the MAPEC Study and Hygia Project.

Timing the anti-hypertensive, anti-inflammatory and lipid-lowering therapies to circadian rhythms is a hot topic of precision or personal medicine. Further investigation is needed to further the understanding of circadian rhythm-driven and modulated mechanisms and processes of atherosclerosis, causal risk factors (such as hypertension, inflammation, hyperglycemia, and hypercholesterolemia), and their chronotherapeutic management. The contents of this article are intended to inform both future basic research and translational medical applications directed at reducing the community burden of the ancient disease of atherosclerosis that is epidemic today. The possible role of circadian clock deregulation, defect, or imbalance, which could result in disruption of the 24 h time structure and promote hypercholesterolemia, hyperglycemia, diabetes, hypertension, obesity, and OSA was not discussed but merits consideration. Proper chronotherapeutic scheduling of hypertension, ASA, and cholesterol therapies to circadian rhythms highlights a simple and low-cost opportunity to improve patient outcomes. Methods and tools, such as circadian diaries and wrist actigraphy, are useful to identify surrogate markers of circadian time that provide precise and personalized guidance to accurately derive the asleep and wake SBP and SBP means and SBP sleep-time relative decline (BP dipper phenotype) from data obtained by ABPM as well as to optimally schedule medications. While such tools and methods are currently not in widespread clinical use, it is an area of rapid evolution. Future technological developments are expected to simplify the translation of basic and proof-of-concept clinical chronobiology research findings into routine patient care. Currently there are several developments in technology, including: (a) a first-generation easy-to-use, low-cost, clinical-grade, wrist-wearable device (Nightview: Omron Healthcare) that makes possible on-demand measurement of SBP and DBP during wakefulness and preprogrammed automatic measurements at three specific times during sleep; (b) on-demand clinical grade ECG assessment instrumentation (e.g., KardiacMobile, AliveCor) that enables screening for bradycardia, tachycardia, and atrial fibrillation; and (c) various high-quality clinical grade wrist wearables useful for the screening of sleep disorders (e.g., Motionlogger Actigraph: Ambulatory Monitoring Inc.; Actiwatch: Phillips).

Taken together, successful mitigation and management of risk factors and key biological processes of atherosclerotic and hypertensive vascular diseases may be achieved by chronotherapeutic control of the atherogenic/hypertensive pathological sequelae through the following steps:

1. Optimal circadian timing, i.e., at-bedtime, of angiotensin receptor blocker (ARB) and angiotensin converting enzyme inhibitor (ACEI) medications to significantly moderate atherogenic actions of angiotensin II when bound to its angiotensin type 1 receptor;

2. Before bedtime ingestion of low-dose aspirin (acetylsalicylic acid) to best moderate platelet aggregation and thrombosis formation that leads to blood vessel narrowing and occlusion;

3. Evening/before bedtime dosing of cholesterol-lowering (e.g., statins) therapy to reduce circulating low-density lipid concentration, a key component of plaque formation and growth;

4. Sleep-time application of continuous positive pressure (CPAP) to moderate OSA-induced pro-atherogenic processes, including elevated asleep BP, plus at-bedtime ingestion of ARB or ACEI hypertension medications to retard, or even regress, the vascular and organ pathology of atherosclerosis in regular patients, and CPAP treated OSA patients whose elevated BP remains uncontrolled.