Review: Circadian clocks and rhythms in the vascular tree

Abstract

The progression of vascular disease is influenced by many factors including aging, gender, diet, hypertension, and poor sleep. The intrinsic vascular circadian clock and the timing it imparts on the vasculature both conditions and is conditioned by all these variables. Circadian rhythms and their molecular components are rhythmically cycling in each endothelial cell, smooth muscle cell, in each artery, arteriole, vein, venule, and capillary. New research continues to tackle how circadian clocks act in the vasculature, describing influences in experimental and human disease, identifying potential target genes, compensatory molecules, that ultimately reveal a complexity that is vascular bed-specific, cell-type specific, and even single cell specific. Though we are yet to achieve a complete understanding, here we survey recent observations that are shedding more light on the nature of the interaction between circadian rhythms and the vascular system with implications for blood vessel disease.

Introduction

Circadian Rhythmicity: Biological Role and Significance

In today’s ‘smart’ households, there is a robo/ecosystem of lights, alarms, thermostats, and even coffee makers that ‘artificially’ anticipates our departures, our arrivals, and other daily activities so that life can be more structured, convenient, and efficient. Efficiency and health intersect, with other technology applications that more specifically anticipate our sleep patterns and activity cycles; our phones dim in the night and gradually brighten in the morning. Technology is being tuned to improve our health. That circadian rhythms and biology have piqued the interest and innovation of the high tech industry is evidence of its relatability/importance and its pervasive impact to human health. The inspiration for these pattern-mechanized devices are our internal biological clocks that themselves are genetically and molecularly mechanized for preparedness; plants that stretch in expectation of light; cells that consume energy when energy is needed; DNA that replicates at 24 hour intervals.[1] These routines of biology continue even when the light isn’t there, a trademark of autonomously-acting circadian rhythms. Anticipation in biological circadian rhythms serves to optimize the efficiency of our internal body systems so that we get things done, at maximal output (or more likely functional output) and minimal input. However, the human condition, biology, and routine are prone to error, and when these routines of sleep and rest rhythms become misaligned with the 24 hour clock, things can go astray. In the long term, clocks may adapt to the loss of routine, and then act to alter regulation of target genes, but this long-term drift by the clock and its outputs from the natural rhythm can eventually devolve into disease, in particular in the cardiovascular system. These shifts may stem from environmental disruptions,[2] as occur in chronic conditions of shift work,[3] sleep loss, alcohol/drug dependence,[4] and even night eating syndrome, [5] or also silently by dysfunction in internal vascular clocks. In the acute events that are the culmination of years of human disease, time of day correlates with the incidence heart attacks and cardiovascular (CV) events, suggesting that a clock is likely still ticking, but perhaps mis-timed, to incorrectly anticipate other ticks (i.e. circulating rheology mechanisms) in the cardiovascular system, to dispense the heart and vasculature in a state of vulnerability, such as a a condition of constriction when blood flow is needed. Herein, we focus on the research insights into the vascular-centric clock, that could complement (or be complemented by) other recent discussions of clocks, in rest and stress conditions,[6] chronotherapy[7–11], melatonin signaling[12], heart clocks[13] and the big picture of integrating the science to human disease[14].

‘If coronary arteries become vulnerable to occlusion when the intima covering an atherosclerotic plaque is disrupted, the circadian timing of myocardial infarction may result from a variation in the tendency to thrombosis. If the rhythmic processes that drive the circadian rhythm of myocardial-infarction onset can be identified, their modification may delay or prevent the occurrence of infarction.’ [15]

In the excerpt just above from the year 1985, the authors postulated that there was a pattern in myocardial infarction (MI) in human patients, one that exhibited a circadian rhythm, dependent on activity/stress cycles and also dependent on vascular responses. They further postulated that understanding the molecular underpinning of this pattern might offer promise for human health, given their prior observation demonstrating a 3 fold increased incidence of MI at 9 AM versus 11 PM.[16] However, not all heart attacks or strokes occur in the morning; this is a pattern and it may be that there is a summation of ‘uneventful’ morning stresses, that does or does not fall in the morning hours.[17] Whether it is the incidence of stroke, heart attack, or aneurysms, [18] the vasculature plays a key role in these events, both in its ability to respond quickly through vasoconstriction/vasodilation and respond chronically by changing its structure through vascular remodeling. Aside from the actual disease ‘event’, there is also evidence that acute treatment of vascular disease in the surgery unit can be impacted by time of day. In angioplasty procedures success has been described to be worse at night,[19]. In contrast, in a 2018 study in France, patients undergoing aortic valve replacement with severe aortic stenosis, suffered more adverse events when undergoing procedures in the in the morning, and this correlated with an increase in Rev-erbα expression in atrial tissue. The authors extended their observations to mice, using ex vivo Langendorff mouse heart studies, and found that Rev-erbα knockout mice and the administration of Rev-erbα antagonists to mice also improved myocardial injury.[20]* The variability of timing (and misfiring) of clocks in different diseases, organs, and cells may underlie these differences and apparent paradoxes.

Rev-erbα is a positive regulator of mitochondria and skeletal muscle respiration[21] but also a repressor of Bmal1 transcription,[22], hence in the above studies where Rev-erbα was suppressed, Circadian rhythm might be expected to be more robust, as the key clock component Bmal1 ought be increased. Bmal1 comprises an essential half (for the most part) of the transcriptional arm of the circadian clock feedback loop, a driving arm whose constituent components all have the common basic helix-loop helix (bHLH) structural motif. Bmal1 along with Clock (or Npas2) protein form a heterodimer, that also interact with histone acetyltransferases, [23,24] to enable Bmal1/Clock to bind and transcriptionally activate the DNA of the Period (Per 1,2, 3 isoforms) and Cryptochrome (Cry1, Cry2 forms) genes, which comprise the braking force of the feedback loop. The antiphase pattern of gene expression of the Per[25] and Cry genes to Bmal1[26,27] and Clock in 24-hr profiles across cell and tissue types is a robust characteristic of the circadian mechanism[28]; when Bmal1 expression peaks, Per expression is at its lowest point. However, the timing of the Per and Cry are not completely aligned by time of day it seems. Recent studies in cultured transformed U2OS cells suggest that there are 6-hr differences in the phase alignment of the negative limb[29] which was also shown isolated mouse liver nuclei,[30] with Per peaking before Cry. While extrapolating timing and potential relevance of peaks and troughs between in vitro assays and in vivo though the antiphase difference between the limbs is a constant, perhaps navigating meaning into the precise timing of phases and oscillations of core clocks to correlation to functional output oscillations may be tenuous, until reproducibility can be better defined and established, from cell to cell, tissue to tissue, and from in vitro to in vivo. The key however, is that the clock oscillates, and it is unique in that the timing of interaction, transactivation, translation, localization, and degradation innately places each gene/protein in the right place (sub-cellular compartment) at the right time, so that the resultant summation of the effect of their relationship is a 24-hour oscillation.

Target or output genes of the transcriptional arm of the clock may have that same or a similar consensus DNA sequence like repressor arm of the clock, i.e. the Per and Cry genes, a non-coding cis element called the E-box (CACGTG), that is an established motif that can confer circadian regulation[31].** Given the robustness of the circadian clock, and its impact on target genes, alteration of circadian specific components’ gene expression might alter output genes, conceivably without affecting overall circadian clock rhythms.’ In recent work, a mouse model with mutation of the non-coding cis element in Per2 was developed, that may provide an important model of circadian dysfunction where all the circadian clock components remain expressed, but with the introduction of a non-oscillating Per2 gene and protein that impaired oscillations in the circadian clock and target genes.[31] However, it maybe that a short circuit in a clock component, that leaves the clock ticking, may be not be a good thing, and that the robustness, while in the short-term could be protective, in the long-term it could alternatively promote disease, as an inability to adapt to a dysfunction in its mechanism.

The complexity of what makes an E-box in the enhancer region a bona fide (if there is one) clock target, involves DNA sequences surrounding the E-box, which may also depend on chromatin modification to relay opening of a particular gene promoter/enhancer target sequence. That opening, that epigenetic modification, is also subject to circadian variation. Importantly, the clock mechanism is self-sustained. The zeitgebers, such as sunlight and diet, set the timing of organ clocks, while there are also synchronizing factors, that maintain cell clocks in the same phase[32]. Even absent these cues whether in individual cultured cells, or in humans and mice kept devoid of light (or in constant light),[33] the molecular clock components continue their rhythms, but with a gradual drift. In individual cells, that ‘drift’ is a loss of synchrony across a homotypic population of single-cell clocks,[34] or in the organism, a gradual delay in wake time or circadian cycle. As a key characteristic of the intrinsic circadian oscillator, the approach to subject laboratory animals to free running conditions remains an important experimental demonstration of a bona fide circadian rhythm.

As to activities that the clock components have, aside from regulating circadian rhythms, Clock itself has been reported act as an acetylase [35] and histone acetyltransferase/HAT.[36] Clock’s partner Bmal1 may also have non-transcriptional activities as a translation initiation factor, by forming cytosolic protein-protein interactions with the eIF translation factors,[37] the latter which facilitate ribosome interactions with mRNA in the ER. These activities, along with their native oscillatory function, comprise a complex network of interaction. Profiling studies reveal that there are many genes that exhibit a circadian rhythm, potential outputs of the circadian clock. Regulation of clock targets may be in part E-box dependent and in part not, primarily regulated or perhaps secondarily regulated, and so-on with the integration of multiple molecular events and physiological responses coordinating into a clock output response.

The vascular clock.

Circadian rhythms in vasomotor vascular function (the acute contractile or relaxant response in blood vessels) occurs in humans.[38] Mutation of numerous components of the circadian clock in the vasculature of rodents,[39,40], validated that the clock was important in vascular function mice with genetic disruption of Bmal1, Clock, and Per, exhibiting endothelial dysfunction,[41] smooth muscle dysfunction,[42] blood pressure impairments,[43], transplantation failure[44,45] and pathological remodeling of the blood vessels.[46] Globally broken clocks resulted in a ‘short-circuiting’ the regulation of molecules important in endothelial signaling \ (nitric oxide, angiotensin, endothelin) and endothelial structure/integrity (Icam1, Claudin-5) and smooth muscle function. In the blood vessels, clock rhythms may integrate across the different cell layers, with paracrine mediators like nitric oxide both under clock control and controlling the clock.[47] While the blood vessels function as a unit to contract, relax, or remodel, this is the result of an integration across[48,49]* and within the smooth muscle[50] and endothelial layers[51]. Whether the smooth muscle cell (SMC) clock functions similarly to the endothelial cell (EC) clock, whether ECs and SMCs are in phase, whether there are EC and SMC bHLH’s that cell specifically regulate the layers are still not well elucidated. In other cell types, recent data points to a number of factors (aside from other interacting proteins) that confer cell clock specificity, including number and location of eboxes, and a novel network of gene enhancer-enhancer interactions.[52]** Other recent work points to other transcription factors that may preserve rhythms in the Bmal1-KO such as the ETS (E26 transformation-specific) transcription factors in liver[52] adding to mechanisms that confer oscillations in the absent of certain core clock proteins, with each organ/tissue perhaps invoking specific adaptation, such as intercellular coupling in neuronal cells.[53] A challenge in analyzing the vasculature and its circadian rhythm, which has typically been at the at the level of the large mouse arteries (aorta, never mind the microsvessels) has always been that of starting material, especially when it comes to the endothelium, the multiple n number, and multiple time points required to formulate a comprehensive circadian time course with sufficient rigor, for the profiling by endothelial single cell sequencing[54,55].

Circadian rhythm of circulating cells and impact on the vasculature

Former cell specific gene targeting work established an important role for Bmal1 in the endothelium of the femoral artery of mice in regulating thrombogenesis, suggested to be through a potential interaction with platelets.[56] Leukocyte adhesion which is closely associated with platelet aggregation to coordinate thrombotic and inflammatory responses,[57] is also dependent on circadian rhythm,[58], an impact that can differentially affect the vascular tree in its functional and structural heterogeneity. In fact, new evidence demonstrates that there are clock-dependent differences in the ability of blood leukocytes to interact at different levels vascular tree: arteries, arterioles, veins, and venules, an important response in inflammatory conditions. [59]** In these studies, it was demonstrated that arteries (common carotids) and veins (jugular) exhibited day versus night differences in leukocyte adhesion, but the phase (time at which peak expression occurred of adhesion) was antiphase with adhesion peaking at night in veins and in the morning in mouse arteries, a rhythm that seemed to be independent of a hemodynamic rhythm and dependent on the clock, but independent of the phase of the core clock (Per2-Luc studies), and the same was found in arteriole and venule preparations from the cremaster muscle. Leukocyte Bmal1 was shown to be key in this rhythm, as Lyz2Cre-Bmal1flox/flox mice lost rhythmic adhesion, with an increased adhesion at the trough point acting to abolish the rhythm. The endothelium seemed to play a specific role in regulating the venous adhesion rhythm, as Cdh5CretERt2 Bmal1 floxed mice lost the rhythm, though arteries rhythms persisted. Circadian targets implicated in the circadian regulation of leukocyte binding based on rhythmic expression patterns in the vessel tissue included adhesion molecules (ICAM-1) and chemokines (Cxcls). Finally, in this comprehensive body of work, the authors suggested that sympathetic denervation chemically and surgically abolished carotid arteries and venous rhythms in leukocyte adhesion. In addition, there are many targets oscillating in the vascular tissue, that likely reflect oscillations in the vascular smooth muscle cell, which is the most prominent cell type. Capillaries, which are the ‘finest’ of vessels being essentially just an endothelial cell layer, have also been implicated to be affected by the clock. Bmal1 disruption decreases capillary organizational formation by affecting the retinal endothelial cells in the cultured bioassay [60], with possible implications for vision and diabetes, while, in heart, capillaries were reported to be reduced in Per2 mutant mice in the infarct zone in the coronary artery ligation model after 4 days.[61] Despite these findings, there are limits in the available tools and approaches to help us study the capillary system, but that the capillary clock is potentially important, may provide further support for the circadian clock’s importance to the endothelium, which in essence is a tube of endothelium.

Recent observations describing the impact of circadian rhythm on atherosclerosis

There is evidence that connects the clock control of circulating cells, and specifically myeloid/monocyte cell rhythms contributes to the chronic (~2 to 3 months) development of atherosclerotic disease, though the outcome seems to be model-dependent. In the apoE-KO mice crossed to LysM targeted Bmal1 mice, atherosclerosis was increased due to increased recruitment of monocytes as evidence in the lesion by flow cytometric sorting of the aorta gated to CD45+CD11b+F4.80 [62] In another set of studies in the apoE-KO mice, circadian rhythm was environmentally disturbed, by altering the light cycle to constant light for 12 weeks. This abolished locomotor rhythm, and increased atherosclerosis in male apoE-KO mice, but not female mice as assessed by aortic root lesion staining.[63]* In a different genetic model of atherosclerosis, the ApoE*3-Leiden CETP mice were exposed to a light cycle regimen where the normal light cycle was switched to a reverse light cycle, every week, for 15 weeks; this also caused increased atherosclerosis, however in those same studies phase advance or phase delay light cycles, in contrast to reverse light cycling, were not found to affect lesion.[64]* This was surprising in that, in general, the jet-lag (phase delay/advance) model of light cycle derangement with its repetitive delays or advances is perceived as a robustly disruptive model, as the cycle is repetitively changed. However, it is possible that the duration of altered light cycle paradigms shifts and advances play a factor. While there may be an initial stress and circadian dysfunction in the early weeks of light cycle derangement, after longer duration, in the case of the this study, 3 months or 12 weeks, the phase delay models result in adaptation. In contrast, the reverse light cycling remained more robust in its ability to disrupt rhythm and induce atherosclerosis. Indeed there are numerous light cycle models,[65] and a take home is that each model needs to be characterized completely, and aside from demonstrating loss of locomotor rhythms, the effect on clock oscillatons on the relevant tissue (i.e. aorta) should be also assessed. In the reverse light cycle study where atherosclerosis was increased in the ApoE*3-Leiden CETP, composition of plaques correlated with increased macrophages. There was also a trend toward a reduction in collagen in the aortic root. Collagen deposition in the fibrotic cap of the lipid lesion in the lumen of arteries is thought to confers a stability of the plaque from rupture, but when thinned results in a vulnerability of the plaque to rupture and occlude downstream microvessels.[66–68] Thus, in those studies the reduced collagen was interpreted to mean a potential of plaque rupture vulnerability. The impact of collagen in itself is complex, depending on the affected disease. While, the fibrotic plaque and the increase in collagen is a good player in atherosclerosis by stabilizing plaques, in the absence of atherosclerotic lesion, increased collagen in the medial/smooth muscle layer has been found be a bad player in the vasculature by impairing its elasticity, a stiffening that tracks linearly with human aging[69], and a stiffening that is more often found in Bmal1-KO and Per-KO mice that WT mice (non-atherosclerotic).[46,70] Thus, depending on context, collagen can both provide structure and stability to fasten a plaque to the endothelium, and that same characteristic of structure and stability when excessively deposited collagen is found in the vascular media absent lesion, as occurs during aging, it contributes to stiffening and loss of recoil capacity. ‘Context’ may also be important for clocks in this way also. For example, as earlier discussed, DD and LL are free running conditions and a defining characteristic of endogenous circadian rhythmicity based on historical observations of plants’ native rhythms[71] observed in darkness[72]. Because circadian rhythms are preserved in free-running light cycle conditions in WT mice with progressive phase delays over time relative to standard 12 hours light/12 hours dark (LD) (aside from the known circadian clock mutant mice [17]), it might be expected that they are not toxic to health. However, the standard free running conditions of 24 hour light cycles (LL) or 24 hour dark cycles (DD) in the long-term (weeks to months) have also been shown to cause impairment in circadian[73] and other cardiovascular[74] responses in animal models. It may be that the summation of temporal phase delays over time may impact overall synchrony of rhythms in the populations cell clocks [34,73] and network dynamics do coordinate the cell autonomous rhythms[75,76]. While the aforementioined reports pointed to a protective role for circadian rhythm in atherosclerotic disease, whereby genetic disruption was deleterious, [62,77,78] there are reports of an opposite effect. In another double knockout model of atherosclerosis where the myeloid Bmal1 was again disrupted but this time in the LDL-receptor knockout mouse, disruption of Bmal1 reduced the atherosclerotic plaque.[79] Consistent with the latter, and supporting the concept that Bmal1 promotes macrophage motility and function to promote disease, in pneumonia induced in mice, Bmal1-KO LysM-Cre mice exhibited enhanced macrophage function and were protected against the infection, with authors concluding that Bmal1 was a repressor of phagocyte responses of the immune system.[80]* The concept of broken clocks being protective is counter-intuitive if not controversial, but has been formerly observed in the context of glucose homeostasis[81] and more recently discussed,[82] with reference to recent findings showing inducible Bmal1-KO’s are protected from other cardiovascular [83] and even locomotor contexts [84],

So what can we make of these disparate effects, and what does it mean relative to the vasculature? The vasculature is not the same across the circulatory system, nor within local segments of the same vessel, i.e. differing in expression patterns across the endothelial cells of the descending aorta[85] or differing in lesion development between the convexity (upper curvature) concavity (lower)[86,87] and the anteriority or posteriority[88] of the aortic arch. Of course, circadian clocks act to regulate numerous targets and their dysfunction results in compensatory or pathological molecular adaptations, which might relate to their individual activities or through the circadian clock pathway in different cell types differently. The integration of a defective clock across endothelial cells, macrophages, smooth muscles cells, liver cells, may reflect varying molecular responses in each tissue. And as previously discussed, the variation in response that Clock or Bmal1 for example, target different mechanisms in atherosclerosis [89] And even the atherosclerosis models, the LDL-r model versus the apoE are mechanistically different models, [90] perhaps the former being more proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent [91] which might have different signaling ramification dependent on the circadian clock mutant studied. PCSK9 has been associated with sleep dysfunction as well as being a target for lipid lowering in humans via the drug evolocumab.[92] In this study of long and short sleeping individuals, a multi-ancestry genome-wide sleep-SNP analysis identified apoliproteins (HDL-c, LDL-c and triglycerides) that associated with long or short total sleep times and further identified associations with SNPs in lipoprotein lipase/LPL and the LDL-receptor regulator PCSK9, though there were no apparent associations with the core clock.[93] **

A Bmal1-knockout rat and the aging clock

Recent work has also been done with the development of a Bmal1-knockout rat, and how this model behaves with regard to blood pressure. While a sex difference in salt excretion in male versus female rats, with males seeming to have a salt regulation that is more Bmal1-dependent,[94] blood pressure and locomotor rhythm were not changed significantly, different from the mouse knockout, though the knockout rat did lose circadian clock gene expression in kidney cortex and had reduced body weight.[95]** These differences observed in the rat Bmal1-KO model may provide additional insight into compensatory mechanisms are involved and whether free running conditions can elicit rhythmic aberrations, or if there are additional complexities involved in the rat model. Importantly, the development of the Bmal1-KO rat model will be useful in experimental disease models that are better suited in the rat.[96,97] Aging is another important factor, that has been previously demonstrated to alter circadian clocks in the aorta of mice, and re-emerged in a recent Rna-seq study revealing a differential impact core clock genes’ expression at single time-points in 3 versus 18 month old mice.[98]* Another study assessed thrombogenicity in young versus older Bmal1-knockout mice and potential associations with brain tissue fibrinolytic system. Consistent with other studies showing Pai-1 induction, the authors find that and platelets are increased in the Bmal1-KO, and also lymphocytes were particular decreased in the aged Bmal1-KO mice, which might be counterintuitive to the atherosclerotic phenotype described during circadian mutation[62,77,78,99] but likely consistent with observations demonstrating circadian control of lymphocyte trafficking[100]. Still much work remains to be understood with regard to the endothelial cell and the circadian clock, but is rapidly moving forward with optimization of single cell and single layer profiling approaches and technical advances that may shed further insight on the models of cell-specific gene disruption.

Conclusion:

Clock dysfunction and clock timing are vascular bed specific, are cell-type specific, and are regionally variable. At times, and not necessarily times of day, clocks appear protective, and at other times (or conditions), clocks appear to be deleterious. While much of biology and the molecules governing biology seem to follow an upright u-curve, whereby there is a range of optimal health, and too much or too little on the ends of the range can cause negative effects, it may be that the same follows for the circadian clock. This range of safety is likely dynamic, whereby optimal amplitude or period or phase, in this case, circadian rhythm, may change with disease or in different conditions and is dependent on a network of communication of activities outside the body, and within the body, and in particular, between all the vascular cells. Another possibility is that the context of disease impacts circadian rhythm differently. Atherosclerosis (and the experimental model used) or aneurysm impact circadian clocks and interact differently with regard to involved mechanisms, such that circadian clocks can have these diverse actions. Is the disorder more closely that of a venous thrombosis or an arterial occlusion? The work discussed suggests that adhesion of leukocytes in veins and arteries is indeed, night and day, respectively.[59] It is no surprise perhaps, that clocks in the endothelium versus smooth muscle; arteries versus veins; arterioles vs. venules; all are acting differently. Still, challenges and limitations remain; identifying vascular-tree specific promoters that can selectively target gene expression in arteries versus arterioles, for example. There are even limits to what we know about the anatomy of the microvessel network. While we know the named great arteries of which there are about 40, the profiling of the identity of the microvessels is still perhaps considered to be a region of such heterogeneity, that defining them systematically may not be feasible, or is it? Also, is breaking down the vessel to its branches and to its cell types, is that level of defining the biology more precise? Or is it a study of a dis-integrated state that is just less physiologic? The whole is certainly greater than the sum of the parts, and as such, sometimes the ‘descriptive’ science while not as precise, may still offer accuracy in its simplicity.

Where are we then with our understanding of vascular clocks? There is clearly a complex integration which we still do not understand. Prior to the identification of the mouse circadian clock,[101] and prior to gene expression profiling,[102] there was the idea that there was a unique ‘key’ or circulating signal or mechanosensor which relayed biomechanical (pressure/flow) information from the bloodstream to the endothelium, and then to the smooth muscle.[103] The concept of course still holds, but it is more complicated than one molecule. In disease, the toxic signals and/or forces that are upregulated or stimulated during inflammation, hypertension, and atherosclerosis would facilitate entry into/across the endothelium, to promote vascular disease. Now, with the added understanding of circadian rhythms, we appreciate the different blood vessels also anticipate, each having ‘expectations’ at their own timing, dependent on their unique structure, exposed shear force, and geometry, while circulating cells also exhibit their own timing, so that the endothelial vascular barrier is restricted and regulated. Disruption of the circadian rhythm (whether in the timing of endothelium or the timing of circulating cells) may facilitate monocyte extravasation, oxidized LDL entry, and other mediators that aggravate the ‘unprepared’ endothelium, its lamina, and the smooth muscle. The interactions are highly complex and far from understood, but underscore the importance of basic, descriptive, but robust empirical evidence which may not tell the entire story, but begin to define the profile and function of molecular timing in the vasculature which will ultimately refine our understanding and therapy of vascular disease.