A wrinkle in time: circadian biology in pulmonary vascular health and disease

Abstract

An often overlooked element of pulmonary vascular disease is time. Cellular responses to time, which are regulated directly by the core circadian clock, have only recently been elucidated. Despite an extensive collection of data regarding the role of rhythmic contribution to disease pathogenesis (such as systemic hypertension, coronary artery, and renal disease), the roles of key circadian transcription factors in pulmonary hypertension remain understudied. This is despite a large degree of overlap in the pulmonary hypertension and circadian rhythm fields, not only including shared signaling pathways, but also cell-specific effects of the core clock that are known to result in both protective and adverse lung vessel changes. Therefore, the goal of this review is to summarize the current dialogue regarding common pathways in circadian biology, with a specific emphasis on its implications in the progression of pulmonary hypertension. In this work, we emphasize specific proteins involved in the regulation of the core molecular clock while noting the circadian cell-specific changes relevant to vascular remodeling. Finally, we apply this knowledge to the optimization of medical therapy, with a focus on sleep hygiene and the role of chronopharmacology in patients with this disease. In dissecting the unique relationship between time and cellular biology, we aim to provide valuable insight into the practical implications of considering time as a therapeutic variable. Armed with this information, physicians will be positioned to more efficiently use the full four dimensions of patient care, resulting in improved morbidity and mortality of pulmonary hypertension patients.

INTRODUCTION

“I don’t understand it any more than you do, but one thing I’ve learned is that you don’t have to understand things for them to be.”

—Madeleine L’Engle, A Wrinkle in Time

Time is fiction, as conceived in the classic young adult novel, A Wrinkle in Time, by author Madeleine L’Engle. In the book, a young protagonist must traverse both space and time in an effort to prevent a faceless and cold being from taking her family; an apt metaphor for the constant struggle between human health and disease. Remarkably, we have only just begun to elucidate the detailed workings of our own internal cellular clocks. The knowledge gained to date has allowed us to advance into an era where we can now develop strategies to beneficially manipulate our own internal clock, including to prevent and treat various maladies (1). Similar to the aforementioned quote, however, the journey of discovery regarding how to apply novel principles of circadian biology to medicine has been complex and fraught with modest applications. This is not unexpected regarding a concept as existential as the molecular core clock, which evolved as it did primordially from the simple orbit of our planet around the Sun.

Pulmonary Hypertension on the Clock

To date, research into the circadian rhythm (for a list of defined clock terminology, please refer to Table 1) has shed light on how diurnal rhythmicity is involved in the pathogenesis of a number of illnesses including cancer (2), Alzheimer’s disease (3), and cardiovascular disease (4). Likewise, the circadian clock has a well-described role in chronic airway disease progression, particularly related to tobacco smoke exposure (5, 6). However, circadian rhythm disruption has not been linked to the pulmonary vascular remodeling resulting in pulmonary hypertension (PH), except indirectly through changes in pressure related to obstructive sleep apnea (OSA) (7), which are classified under a broader heading of World Health Organization (WHO) Group 3 PH.

Table 1.

Circadian terminology

	Definition and Explanation
Circadian rhythm	An approximately 24-h oscillation that persists in constant conditions, influenced by external cues.
Period	Time from peak to peak of circadian oscillatory factor being measured.
Amplitude	Difference between the peak and mean values of circadian oscillatory factor.
Acrophase	Timing of the peak value of monitored parameter, such as leukocyte count.
Zeitgeber time (ZT)	(German for “time giver”) a stimulus that cues time signaling, such as light or nutritional intake. ZT0 represents that onset of stimulus.
Circadian time (CT)	A superior reflection of true organismal timing in the absence of external stimulus (such as light). CT0 would thus be the start of a subjective day, when the lights would have been on, for example.

CT, Circadian time; ZT, Zeitgeber time.

It is perhaps useful to note that circadian-related variation effects on systemic vascular remodeling and hypertension, which can contribute to cardiovascular disease, have been described for decades (8). Unfortunately, little is known regarding circadian variability contributing directly to PH, despite interest in whole lung effects (9). This may be in part due to the changing landscape of the clinical and research fields. PH has recently been redefined by the 6th World Symposium on PH (WSPH) as pulmonary vascular resistance (PVR) greater than or equal to 3 Wood units (WU). Additional criteria include all forms of precapillary PH associated with a mean pulmonary artery pressure (mPAP) of greater than 20 mmHg, different from the previous definition of greater than 25 mmHg (10). Although this excludes PH due to left heart disease [WHO Group 2, with pulmonary artery occlusion pressure (PAOP) greater than 15 mmHg], the definition continues to apply to a broad swathe of those with pulmonary vasculopathy related to various conditions, including pulmonary arterial hypertension (PAH; Group 1), chronic thromboembolic PH (CTEPH; Group 4), or multifaceted (Group 5).

As mentioned, PH secondary to chronic lung disease and/or chronic hypoxia-exposure (Group 3) does include some primary data linked to circadian variation, primarily through examination of patients with OSA where there are complications related to either a decrease in circulating oxygen tension or sleep fragmentation. It is noteworthy that the scant amount of literature available on the topic would suggest that increases in nocturnal mPAP in patients with OSA and pre-existing PH is worsened independent of the degree of hypoxia (11) and OSA severity index (12). This suggests that in susceptible people there is at least a potential contributory role of the sleep fragmentation to disease pathology independent of hypoxic events. Supporting this concept is that the severity of OSA itself is associated with aberrant regulation of canonical clock genes in the circulation (13).

At least one animal model of PH—sugen/hypoxia [a combination of vascular endothelial growth factor (VEGF) receptor antagonist and chronic hypoxia exposure]—phenocopies disruption in circadian variability, whereby there are clear changes in the degree of mPAP elevation depending on time of day of measurement in treated animals versus controls (14). Likewise, intermittent hypoxia alone is associated with intertissue misalignment of peripheral organ and cellular clock functions, especially within the lung (15). This mirrors the effects of sustained hypoxia associated with pulmonary vascular remodeling, namely, microvascular rarefaction with associated large vessel proliferation and vessel narrowing. Thus, examining circadian variability in pulmonary vasoconstrictive responses that contribute to long-term changes in pulmonary vascular function is a potential area of interest to the field (16). This is especially applicable to Group 3 PH, which has limited options available for treatment and is believed to be mediated by long-term permanent adventitial fibrotic accumulation.

Thus, the purpose of the following review is to summarize the current available information regarding the contribution of circadian disruption to lung vascular remodeling and inflammation (17), focusing on the cellular and molecular core clock pathways (refer to Fig. 1) with relevance to PH, creating a wrinkle in pulmonary vascular research, one with ample space and time for drug development outside of current vasodilator-based therapies (18).

Figure 1.

Molecular and cell-specific contributions of the circadian core clock with relevant pulmonary hypertension pathways highlighted. BMAL1, brain and muscle ARNT-like 1; CRY, cryptochrome; EndoMT, endothelial-to-mesenchymal transition; PDGF, platelet-derived growth factor; Per1/2, period 1 and 2; PH, pulmonary hypertension; ROR, RAR-related orphan receptor; RORE, ROR element; TGF-β, transforming growth factor-β.

CELL-SPECIFIC CIRCADIAN CONTRIBUTIONS TO VASCULAR HEALTH AND DISEASE

Given the diverse number of relevant core clock proteins, acting both centrally and within peripheral organs, it is perhaps helpful to discuss first what is known regarding intrinsic clock effects, at the tissue-level, versus circadian effects that are linked to systemic regulation, directly or indirectly related to lung vascular pathology.

Hypothalamus (Suprachiasmatic Nucleus)

The contribution of the hypothalamus to monocrotaline-induced PH in rats was previously described, though the focus was solely on the sympathetic drive associated with direct neuroinflammation (19). Although it is not the only central nervous system contributor to circadian biology (20), the suprachiasmatic nucleus (SCN) located within the hypothalamus serves as the central governor of organismal circadian rhythm (21) and is entrained to light (22). Importantly, ablation of the SCN does not lead to complete loss of lung circadian biology, just asynchrony between other tissues (23). Recently, native lung cells have been described as a direct interface between the central SCN and the peripheral lung circadian biology (24) due to their coordination of circadian entrainment.

It is noteworthy that many pathologic vascular clock effects described in the periphery are negligible so long as SCN signaling is preserved (25), lending support to the SCN role as a master vascular circadian pacemaker. Furthermore, this effect appears independent of vasodilatory pathway influence through either endothelial nitric oxide synthase (eNOS) (26) or neuronal nitric oxide synthase (nNOS) (27). However, there does appear to be a role for peripheral tissue level expression of clock genes in maintenance of systemic vascular health (28), summarized recently (29). Still, there are compelling human data that associate changes in SCN activity, primarily defined as changes in the vasopressin signaling axis effect on peripheral dopamine and epinephrine release, with resulting elevated systemic blood pressure (30). It is unclear if this is a cause or an effect, however, as systemic hypertension can cause independent changes to SCN activity itself. The role in PH is, to date, undescribed.

Smooth Muscle Cell

Intriguingly, one of the first cell types in which peripheral circadian clock regulation was described (31) was smooth muscle cell NPAS2-mediated change in Period 2 (Per2; a clock “inhibitor”) rhythmicity, particularly prevalent in the vasculature (32), although easily modeled in vitro (33). This phenomenon is influenced by, but not dependent on, SCN governance through adrenergic signaling (25), and the angiotensin-signaling pathway (34, 35). In particular, chronic changes in vascular wall elastance/stiffness due to myofibroblast proliferation and differentiation is influenced by circadian regulation of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9), with core clock transcription factor Brain and Muscle ARNT-Like 1 (BMAL1) knockout (KO) and related Per1/2/3 KO mice displaying pathologic remodeling of large vessels (36).

In aging mice in particular, rhythmic disruptions can heighten cellular senescence, which is reinforced by decreased telomerase activity, resulting in a reduced ability of cells to transmit circadian signals to the core clock machinery (37). Ultimately, this decreased response to external stimuli in smooth muscle cells has consequences with respect to propensity for atherosclerotic plaque formation and myocardial infarction (38), without described pulmonary effects. These data are consistent with the known role for BMAL1 in regulating platelet-derived growth factor B (PDGF-B) signaling (39), a main driver of smooth muscle cell proliferation (40), which is also linked to PH. This observation may, in part, underly the observation that deletion of BMAL1 from smooth muscle cells is actually protective against the development of aortic aneurysm and rupture, through suppression of MMP family enzymes (41). Likewise, BMAL1 transcription cofactor CLOCK gene expression in vascular smooth muscle cells is a critical mediator of autophagy related to atheromatous plaque formation (42). Similar to recent work in PH (refer to circadian therapeutic implications), this phenomenon is wholly reversed by administration of rapamycin. More work remains to be done, however, to clarify protective or deleterious roles for other clock function in this cell type, related to pulmonary disease in particular.

Endothelial Cell

Of the described tissues and cell types, circadian variation in endothelial cells is most correlative with vascular disease and maintaining homeostatic function in healthy adults (43). The circadian clock also plays a large role in developmental angiogenesis (44), reviewed recently (45). In endothelial cells, a pathogenic change that manifests due to the alteration in circadian rhythmicity is the decreased expression of eNOS, which is typically associated with decreased endothelial health, particularly in aging patients (46). These correlative findings are reinforced by further mechanistic studies in Per2 mutant mice, whereby endothelial contraction is altered, although not necessarily associated with a hypertensive phenotype (47). However, there is an associated decrease in endothelial cell proliferation, VEGF receptor expression, and increased profibrotic milieu and vascular damage (48, 49) in similar models of disease.

In particular, vascular endothelial cell expression of thrombomodulin is heavily regulated by the circadian clock (50). Not coincidentally, thrombomodulin also serves as a surrogate biomarker in PH (51), especially in systemic sclerosis (SSC) related disease (52), with improvements in blood levels of protein resulting from treatment with prostacyclin therapy (52). Thrombomodulin’s role in promoting a fibrotic phenotype may explain the increased clotting within BMAL1 KO mice, which is also contributed to by the release of von Willebrand factor (vWF) from endothelial cells (53). BMAL1 KO mice display uncoupling of eNOS, associated with maintenance of endothelial cell barrier function (54), although this is unlikely to by itself be sufficient for the induction of PH (55). However, at least one group has demonstrated that in an endothelial cell-specific model of BMAL1 deletion—also described in “jet-lagged” mice—nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation occurs through compensatory unopposed CLOCK-protein activation (56). More recently, a similar model of endothelial-cell specific deletion of BMAL1 (using a Tie2 Cre-recombinase construct) was shown to actually decrease the burden of atherosclerotic disease (56), although the inflammatory milieu within the vessel was not fully characterized. A similar endothelial cell pathogenic state was seen in a model of increased CLOCK expression, followed by in vitro hypoxia exposure, leading to inflammatory cascade signaling (57). This is ultimately unsurprising given the high degree of overlap between clock and hypoxia-inducible factor pathway activation and homology (58), as recently described. The similarity may also explain shared roles of both clock and hypoxia-signaling in vascular senescence, whereby Per2 mutant mice have impaired ischemia-induced vascularization through decreased endothelial cell (EC) mobilization and function (59).

Endothelial-to-mesenchymal transition (EndoMT) describes the alteration in phenotype of endothelial cells to α-smooth actin (α-sma)-producing mesenchymal cells, known to contribute to stromal remodeling and fibrosis in cancer, chronic tissue scarring, and pulmonary vascular disease. In PH, EndoMT contributes to worsening disease via regulation of the BMP receptor 2 (BMPR2), a canonical genetic contribution to PAH development (60). Interestingly, BMAL1 KO mice display an increase in BMP-dependent EndoMT (61) through BMP9, previously implicated in PAH pathogenesis (62). Given that EndoMT is also worsened by CLOCK deficiency, in a model of atherosclerotic disease, further exploration of this contributing factor in circadian-mediated disease appears prudent.

Myeloid Cell

As a whole organism model of circadian disruption, sleep fragmentation is prominently associated with an increase in circulating leukocytes. In particular, monocytes have been demonstrated to increase invasive capability in patients with severe obstructive sleep apnea, although this is mainly believed to correlate with degree of hypoxemia (63). There appears to be more than just a hypoxic effect on cells though, as decreased REV-ERBα also directly inhibits the inflammatory function of macrophages through a decrease in CCL2 expression (64). This ultimately leads to a decrease in cell adhesion and migration, associated with multiple chronic inflammatory diseases. Likewise, BMAL1 and Per1/2/3-signaling is associated with inhibition of the robust adaptive and innate immune cell inflammatory cascade related to transplant graft vasculopathy development (28).

An additional clue to the mechanism of myeloid cell release and activity lies in the study of circadian influence on neutrophil adherence in response to sterile and nonsterile injury (65, 66). Innate immune cell peripheral clocks drive migration and compartmentalization of neutrophils in particular, regulated by BMAL1-induced expression of CXCL2 and subsequent increase in autocrine and paracrine CXCR2-signaling (67). The expression pattern favors homeostatic egress of myeloid cells into the tissue at night (the murine active period), increasing antimicrobial activity and subsequently stimulating vascular inflammation and death, as these cells age. Thus, an “aged” neutrophil phenotype has been defined over the course of a single-day, whereby circadian rhythmicity is known to 1) inhibit the granule content of polymorphonuclear leukocytes, 2) inhibit the capability of neutrophil extracellular trap (NET) formation, and 3) contribute to the development of severe acute respiratory distress syndrome in human patients (68). These phenomena have also been associated with CXCL2/CXCR2 signaling in the BMAL1 KO mice model.

Myeloid cell-specific BMAL1 deletion also highlights the role of Ly6C^hi monocytes in various disease states. The number of these active (CCR2^hi) cells increase during the beginning of the animal active phase, which is reflected in tissue migration after sterile injury (myocardial infarction), dependent upon the time of day (62). An additional consequence is that in LysM.Cre-BMAL1^fl/fl mice, with relatively myeloid-specific deletion of BMAL1 there is increased recruitment of Ly6C^hi monocytes, leading to worsening atherosclerotic plaque development in susceptible animals (69). The same mouse model has also been shown to retard atherogenesis and aneurysmal formation, associated with globally decreased inflammatory markers (70). There is an immunologic balance to core clock protein expression, however, as the same model increases susceptibility to sepsis-related death (71). The effect is purported to be primarily due to circadian effect on mitochondrial function and reactive oxygen species (ROS) generation (72), another common pathologic link to PH.

Finally, sleep fragmentation alone is capable of inducing comparable changes to the myelopoietic compartment, with an increase in a similar population of Ly6C^hi monocytes exacerbating sterile myocardial infarction in response to interrupted sleep (73). The effect could, in part, be due to specific release of myeloid cells with immunosuppressive capabilities, so called myeloid-derived suppressor cells (MDSC), in response to sleep fragmentation. Supporting data for this assertion is slight, but MDSCs have been demonstrated to play a role in models of chronic inflammation, including multiple sclerosis (74) and arthritis (75). Our own group has previously shown this cell population to be relevant to the pathogenesis of PH in several models of disease (76–78).

MOLECULAR CORE CLOCK IN VASCULAR HEALTH AND DISEASE

To discuss the application of circadian biology to the field of PH, we must first define the core circadian clock machinery in relation to a variety of PH relevant molecular signaling pathways. First, it is important to note that the molecular clock mechanism underlying the ∼24 h timekeeping was originally discovered to be a transcriptional-translational feedback loop, with the components of the core clock being ubiquitous across all cell types. Their function has been detailed in many focused reviews to date (79).

Brain and Muscle ARNT-Like 1

Brain and muscle ARNT-Like 1 (BMAL1) is the quintessential circadian transcription factor which functions upon binding as a heterodimer with circadian locomotor output cycles kaput (CLOCK) protein, described in more detail in the following section, to generate circadian function (80) in peripheral tissue (81) as well as the governing central clock in the SCN of the hypothalamus (82). Fortunately, the essential role of BMAL1 to maintaining vascular endothelial health is known; in the absence of the protein (BMAL1^−/− mice), animals experience constitutive uncoupling of endothelial nitric oxide synthase, decreased nitric oxide availability, and subsequent endothelial dysfunction (54). However, the damage incurred can be corrected with supplemental antioxidant treatment alone. This is interesting in light of the known detrimental effect regarding pulmonary BMAL1 inhibition in response to noxious stimuli, such as cigarette smoke contributing to the development of emphysema and chronic obstructive pulmonary disease (COPD) (83). On the opposite end of the spectrum, BMAL1 has also been shown to be necessary for profibrotic transforming growth factor-β (TGF-β)-mediated pulmonary fibrosis (84). These findings are relevant as both COPD and pulmonary fibrosis are major contributors to Group 3 PH, with patients having significantly decreased lifespans compared with those without pulmonary vascular remodeling, and currently no available disease specific treatments (85). Based on differing roles in emphysematous versus interstitial lung disease, the effect of BMAL1 in PH is unlikely to be straightforward.

Global BMAL1 deficiency results in premature aging and a decreased lifespan, regulated primarily through an increase in mammalian target of rapamycin (mTOR) signaling, and therefore partially reversed by administration of rapamycin (86) to affected animals. Importantly, mTOR has recently been heavily implicated in the pathogenesis of PH (87), specifically with mTOR activation contributing to an emphysematous PH phenotype in one study (88) and mTOR inhibition leading to a reverse in right ventricular remodeling in rats with PH (89).

Although it is known that lung tissue in particular is sensitive to circadian disruption compared with other rhythmic organs such as skeletal muscle (90), there is strong evidence that at the cellular level, rhythmic lung inflammation is due predominantly to shifts intrinsic to pulmonary leukocytes, dependent primarily upon BMAL1-expression and activity (91). Consistent with the described relationship between BMAL1 and metabolically important protein signaling pathways (mTOR and sirtuins), myeloid cells in BMAL1^−/− mice display an elevation in hypoxia-inducible factor (HIF) stabilization—and thus hypoxia-response element signaling—due to downregulation of HIF proteolysis (92). HIF, a transcription factor canonically presiding over the tissue and organismal response to low oxygen, has previously been implicated in the pathogenesis of PH (93). Interestingly, as recently reviewed in the American Journal of Physiology-Cell Physiology, the connection between hypoxia-response through HIF and circadian rhythm goes beyond the equally important evolutionary pressures of oxygen handling and light/day cycle; HIF can interact directly with the target of the circadian gene promoter known as E-box, directly affecting cellular rhythmicity (94). It is tempting, therefore, to hypothesize a link between this circadian dysregulation and the tissue-specific HIF contribution to PH development (93). However, a more in-depth core clock examination is needed in these models of disease to make this connection.

The question then becomes: if BMAL1 deficiency is potentially deleterious in PH, is BMAL1 overexpression a salutatory target for potential prevention or treatment of disease? One may look to circadian effects on the TGF-β signaling super-family for clues to the answer, of which BMPR2 is a key member. BMPR2 has been well-described as the most common genetic mutation associated with familial pulmonary arterial hypertension (PAH) (95, 96). Although the exact pathophysiologic mechanism underlying BMP-contribution to PH development has not yet been fully described, at least a significant component is thought to be related to BMP-ligand regulation of endothelial cell-specific apoptosis (97). Interestingly, active BMAL1 rescue is associated with a decrease in endothelial cell expression of bone morphogenetic protein (BMP) related proteins [BMP2, 4, 9, and inhibitor of differentiation 1 (ID1)] (61). In particular, BMP9 has been demonstrated to increase the degree of experimental PH, with BMP9-inhibition yielding protection against development of pulmonary vasculopathy (98). More studies are required, however, given that other groups have drawn the opposite conclusion, finding that BMP9 is therapeutic in the treatment of PH (97). BMAL1, however, remains a viable upstream mediator to study.

Circadian Locomotor Output Cycles Kaput

As mentioned above, circadian locomotor output cycles kaput (CLOCK) is a similar transcription factor partner to BMAL1 in that it is also associated with HIF1-α accumulation in vascular remodeling studies (99), as well as models of atherosclerotic plaque formation. In the latter example, CLOCK overexpression leads to the expression of hypoxia-response element (HRE) gene plasminogen activator inhibitor-1 (PAI-1), leading to coronary artery disease in susceptible mice (100, 101). Interestingly, CLOCK in combination with BMAL1 regulates the expression of SIRT1, which is necessary for homeostatic regulation of cellular metabolism (102). SIRT1 has also been demonstrated to play a protective role in the evolution of PH secondary to either monocrotaline (103) and chronic hypoxia exposure (104). Interestingly, although no direct link has been made to date between CLOCK and PH; however, one study did find evidence of systemic hypertension in CLOCK-mutant mice, associated with loss of function and intrinsic period lengthening (105).

Cryptochrome 1 and 2

Like so much of the existent clock literature, there exists no direct link between circadian clock negative regulators cryptochrome 1 and 2 (CRY1/2) and PH; however, again further insight into pulmonary vascular disease may be gleaned from what is known in the physiological and systemic hypertension literature. First, in patients with severe hypoxia, CRY1 protein concentration in the circulation is known to be decreased, although pulmonary pressures were not assessed in the referenced study (106), despite the knowledge that the protein stains intensely in healthy lung (107). These findings are consistent with another report that showed that haplotype variants in CRY1 were significantly associated with systemic arterial hypertension (108). Intriguingly, CRY1/2-null mice have an increased proclivity to developing salt sensitive hypertension, though it is predominantly mediated through increased aldosterone expression by the adrenal glands (109). However, CRY1 overexpression is conversely associated with protection against the development of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-mediated endothelial cell inflammation due to sleep deprivation-induced vascular inflammation (17).

Finally, CRY1 also acts as a broad histone deacetylase (HDAC) inhibitor, a novel signaling pathway targeted in treatment of various types of PH (110–112). In one study of pulmonary artery-banding in rats, suppression of HDAC worsened right ventricular dysfunction (113). This finding does not rule out a potential compensatory role of HDAC upregulation in certain forms of pulmonary vascular disease, nor does it rule out an undescribed feedback mechanism for Cry1 regulation.

Period 1 and 2

Period 1 and 2 (PER1 and PER2) are a key part of the core timekeeping mechanism upregulating diurnal oscillatory rhythm and are thought to act in large part within the medulla oblongata to regulate, or at least self-sustain, salt-sensitive hypertension (114). In particular, PER1 knockout mice exhibit elevated tonic blood pressure (115), associated with increase circulating levels of endothelin-1 (ET-1) (116), thought to be mediated predominantly through negative feedback on BMAL1 transcriptional activity. Of course, endothelin-axis signaling in particular is a potent mechanism for the pulmonary vasoconstrictive response contributing to PH, and endothelin receptor antagonists (ERAs) are a cornerstone of therapy for patients with Group 1 disease (117). Endothelin has likewise been associated broadly with systemic vascular disease, in particular, the development of end-stage renal disease and associated systemic hypertension, with again—a strong circadian component (118). This circadian role in systemic disease, via PER1 and other core clock proteins, likely contributes to an increase in cardiovascular morbidity in middle-aged adults (119) and warrants potential future investigation regarding its role in PH associated with underlying chronic biventricular heart failure (WHO Group 2 PH, due to left-sided heart disease).

With evidence primarily indicating a role for PER1/2 in regulation of circadian biology, peripheral expression has been noted in the kidney, influencing hypertension. Mice administered PER1 inhibitors displayed decreased sodium channel expression in the kidney and an associated decrease in systemic blood pressure (120). Global Period isoform knockout (Per1/2/3) mice, however, are predisposed to the development of angiotensin-II-mediated hypertension (121), a recently described therapeutic target in treatment of PAH (122). Interestingly, there is evidence that this may be sex-specific phenomena, with female mice being protected against development of PER1-mediated hypertension (123), mirroring the “sex paradox” seen in models of disease; women develop a disproportionate amount of PAH, whereas female mice tend to be resistant to models of disease (124).

Globally, decreased circadian rhythmicity has been linked to decreased nitric oxide (NO) production, associated with decreased Period expression in the elderly (46). A similar finding has also been described at the cellular level, with downregulated Period associated with onset of cellular senescence (37). In particular, PER2 knockout mice display decreased NO production and prostaglandin synthesis associated vasoconstriction (47); a similar alteration in NO synthesis has long been recognized as contributory to development of various forms of PH (125).

Finally, in either BMAL1- or PER1/2-deficient mice, there is an association with increased extracellular matrix stiffness and a decreased vascular compliance, with resulting elevation in commonly implicated remodeling proteins, such as matrix metalloproteinase-2 or -9 (MMP2/9) (126). Likewise, these and associated matrix signaling proteins are undergoing robust experimentation searching for druggable targets in the PH field (127).

Retinoic Acid Receptor-Related Orphan Receptor α and γ

Formerly orphan nuclear receptors, the retinoic acid receptor (RAR)-related orphan receptor (ROR) family encompasses an evolving group of pharmacologic targets heavily involved in circadian biology. Interestingly, these receptors largely function as a positive component of the transcriptional machinery for Bmal1/Arntl (128). RORα/γ are known to control expression of several other pathways discussed in this study, as well as in a variety of cell types (129). Primarily thought to act in a proliferative capacity for Th17 signaling in inflammatory conditions, RORγt levels in the peripheral T cells of whole blood correlated well with the development of connective tissue disease (CTD)-associated PAH in an at-risk population (130). This also corresponds well with what is known regarding the nuclear receptor in the systemic hypertension literature (131), though in both cases expression is thought to be indirectly related to disease pathogenesis. In addition, in at least one model of disease, it would appear that ROR isoforms have little effect on systemic hypertension development in a circadian fashion (132). Adding to this body of evidence is the description of RORγt as working in conjunction with the small ubiquitin-like modifier (SUMO) pathway to influence the stabilization of various proteins marked for ubiquitinylation during hypoxia exposure, including HIF1-α (133). This finding is consistent with similarly uncovered SUMO-regulatory networks, specifically related to HIF, in models of hypoxia-associated PH (134, 135).

Nuclear Factor, Interleukin 3 Regulated

Nuclear factor, interleukin 3 regulated (NFIL3) is a transcription factor that regulates a diverse series of cellular functions, including influence over proliferation and circadian biology. These regulatory roles have made it an exciting target for cancer and age-related disease treatments (136). Of relevance to the circadian field, NFIL3 is a primary driver for Per2 circadian expression (137), which is specifically important in the diurnal development and function of natural killer (NK) cells (138, 139). As previously described, NFIL3^−/− mice (which are deficient in NK cells) develop severe spontaneous PH (140). This phenomenon may be related to the fact that NFIL3 is capable of directly binding to the BMP4 promoter, silencing expression of BMP4 and contributing to the diurnal oscillatory expression of the TGF-β family member (141). Given that mice with heterozygous deficiency in BMP4 (BMP4^+/−) do not develop elevated pulmonary pressures in response to chronic hypoxia exposure (142, 143), it reasonable to speculate that NFIL3 expression is protective against the development of PH.

Rev-ErbA α and β (Nr1d1/2 or REV-ERBα/β)

REV-ERB nuclear receptors play a critical role in circadian biology, primarily through direct inhibition of Bmal1/Arntl transcription (144). This makes the signaling network a prime candidate for druggable targets in advancing pharmacotherapy related to the core clock (128, 145). To date, much work has been done describing the necessary roles of REV-ERBα/β in homeostatic regulation of the pulmonary inflammatory response, primarily through influencing innate immune cells within the lung (146).

In particular, REV-ERBα is known to suppress chemokine CCL2 expression by monocyte/macrophage-lineage cells (64), which are known themselves to be necessary and sufficient for the development of hypoxia-mediated PH (147) through native receptor CCR2 (148). Similarly, REV-ERBα directly—via binding to the IL6 promoter region—and indirectly—through an NF-κB binding motif—inhibits IL-6 signaling (149), which is considered to have a protective effect in patients with PAH (150).

These phenomenon mirror what is known regarding the role of REV-ERBα in a more tissue-specific fashion regarding other forms of vasculopathy. For example, REV-ERBα expression by myeloid cells protects against the development of atherosclerotic plaque formation, with deficiency of the protein resulting in more severe myocardial infarction (151). Thus, more cell-specific studies may be of benefit to elucidate the molecular role of REV-ERB in the development of PH.

CIRCADIAN THERAPEUTIC IMPLICATIONS

A useful conceptual schema for considering circadian therapeutic interventions was recently summarized (152) in the following three strategies: 1) “clocking the drugs” with consideration of not only time of day dosing but also the effects of indicated drugs on the circadian clock; 2) “drugging the clock,” whereby the molecular core clock is directly influenced; and 3) “training the circadian clock,” acknowledging that a significant portion of our rhythmic activity is dictated by behavioral interventions. Borrowing this framework, we attempt to group existing and potential PH therapies below into one of these headings, training our circadian clock either through behavioral interventions, timing of drug administration, or targeting of the core circadian clock molecules that already play a therapeutic role in treating several diseases with shared pathways (refer to Table 2).

Table 2.

Clocking PH drugs and drugging the clock in PH

Medication	Drug Type	Core Clock Target	Core Clock Effect	Time of Day	References
Clocking current and potential PH drugs: how do they influence circadian rhythms?
Imatinib	TKI/PDGFR inhibitor	Unknown/none	Shortened circadian period	Insufficient evidence for speculation	(153, 154)
Sildenafil	PDE5 inhibitor	CREB-responsive genes (Pers and Crys) *indirect	Enhanced phase response/re-entrainment rates	PDE5A gene peaks just before midday – drug after lunch	(155)
Macitentan	Endothelin receptor antagonist	N/A	Not studied	EDNRA gene expression peaks at midday in human lung – drug after lunch	NCT02554903 (156, 157)
Rapamycin	mTOR inhibitor	CRY1 induction is mTOR dependent	Increased Per2 period length, decreased amplitude	Cry1 peaks late in evening – drug before sleep	(89, 158)
Resveratrol	Sirtuin1 activator	BMAL1 and PER2 deacetylation	Shortened behavioral period length in nonhuman primates	Insufficient evidence for speculation	(159–162)
Pioglitazone	PPARγ-agonist	RORE transcriptional regulation *indirect	Not studied (restores blood pressure rhythm in T2DM)	Because this would increase Bmal1 transcription, drug early morning	(163, 164)

Treatment	Drug Type	Core Clock Target	Circadian Effect	Relevance for PH Treatment	References
Drugging the clock: compounds with direct effects on core clock machinery
Nobiletin	Polyphenolic ROR agonist	ROR agonism increases Bmal1 transcription	Increased mRNA oscillatory amplitude. Increased period length.	Wide-ranging metabolic improvements, including reduced inflammation and atherosclerotic development	(165–167)
Retinoic acid (ATRA)	Retinoic acid supplement	E-box containing genes	Phase advance and reduced mRNA oscillating amplitude in liver of mice.	No published positive effects in vivo	(168–171)
KL001	Cryptochrome stabilizer	CRY proteins	Increased period length Decreased amplitude	1) Improved glucose tolerance in obese mice 2) Decreased glioblastoma stem cell proliferation in vitro, and tumor growth in vivo 3) Decreased viral replication of COVID-19 in lung epithelial cells	(172–175)
GSK4112 SR9009/9011 SR8278	Rev-Erb targeting drugs	No current clinical trials exploring use of these drugs in PH
GSK4112	Synthetic compound	Rev-Erb agonist	Phase modulator	Inhibits TGF-β/CS-induced fibroblast differentiation in human fetal lung fibroblasts Inhibits LPS induced pro-inflammatory cytokines release from both small airway epithelial cells and mouse lung fibroblasts	(176,177)
SR9009/9011	Synthetic compound	Rev-Erb agonist	Phase modulator	Reduced cigarette smoke induced inflammatory response + EndoMT	(177)
SR8278	Synthetic compound	Rev-Erb agonist	Phase modulator	Exacerbated LPS-induced lung permeability and increased cellular infiltration	(178)

Intervention	Targets/Effect	Protocol	References
Training the clock: treatments and interventions that strengthen systemic circadian rhythmicity may confer health benefits to individuals with PH
Calorie restriction	Increased lifespan and circadian alignment	30% calorie restriction for 25 wk rescues age-related decline in circadian oscillations of metabolism in liver and stem cells in rodents.	(179,180)
Time-restricted feeding	TRF decreases internal desynchrony between clocks	Food intake (limited to 2 h, 5 h, 8 h, or 12 h), without calorie restriction, prevents weight gain during high-fat diet, and increases circadian rhythms in peripheral tissues in rodents.	(181–183)
Melatonin supplement	Consolidated sleep pattern	10 mg melatonin 1 h before sleep increased sleep latency and sleep consolidation in healthy young men	(184)
Orexin receptor inhibitor		Almorexant (dual orexin receptor antagonist) decreases alertness and accelerates latency to sleep in young men	(185)
Melatonin	Antioxidant/anti-inflammatory	6 mg/kg of melatonin for 4 wk reduced monocrotaline-induced increases in RV systolic and developed pressures in rodents. Melatonin supplementation also blunted MCT-induced markers of oxidative stress in plasma.	(186)
Melatonin + TRF	Strengthening circadian signals	6 wk of melatonin (1 mg) and time-restricted eating in healthy adults.	Active Clinical Trial: NCT03490864
Exercise	Systemic circadian alignment	4 wk of exercise training in mice aligns circadian phase (lung, muscle, brain) to the time of exercise in mice. Exercise may align rhythms in desynchronized individuals	(187)
	Positive effects on PH patients	3 wk in-patient, 12 wk at home with a mix of cycling, walking, light weights.7.3% reduction in pulmonary arterial pressure.19.3% reduction in pulmonary vascular resistance.12.5% increase in cardiac output.	(188–190)

BMAL1, brain and muscle ARNT-like 1; CRY, cryptochrome; EndoMT, endothelial-to-mesenchymal transition; MCT, monocrotaline; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; Per2, period 2; PH, pulmonary hypertension; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; ROR, RAR-related orphan receptor; RORE, ROR element; RV, right ventricular; T2DM, type 2 diabetes mellitus; TGF-β, transforming growth factor-β; TRF, time-restricted feeding.

Clocking the Drugs

Chronotherapy/chronopharmacology.

Chronotherapy is a newly emerging practice designed to deliver therapy at optimal times to maximize effectiveness and decrease associated toxicities, whereby the need to fully chronotype patients is necessary. As an illustration of relevance to lung disease, dose timing of prednisone in patients with asthma was previously demonstrated to be very effective in suppressing symptoms, with an overall decrease in the amount of drug required for maintenance therapy (191). The consideration of timing, in this case, is a sign of an increasing recognition on the role of circadian factors as an integral part of clinical translational research and that end-organ response encompasses core clock biology (192). To date, this accounts for everything from dosing intervals of chemotherapy to appropriate timing of antihypertensive medications (193). There is no reason to doubt that PH medication efficacy would benefit from similar studies given the large overlap between drug targets and circadian mediated genes (194). For example, focus on chronopharmacologic targeting of the CCL2-CCR2 chemokine signaling axis demonstrates that decreasing circulating myeloid cell contribution to atherosclerotic plaque formation can be coordinated to also prevent inhibition of healthy microvascular remodeling, injury repair, and antimicrobial effects of this otherwise salutary cell population (195). Thus, consideration should be given to treatment of PH in the fourth dimension.

Pulmonary hypertension medications.

Phosphodiesterase type 5 inhibitors.

Although many common drugs have been implicated in targeting of circadian genes (194), little is known regarding the effect of currently indicated PH medications on circadian balance. In the case of rodents, change in light-induced phase advances involves activation of guanylate cyclase, resulting in cGMP production (196) (a known mediator of pulmonary vasodilation). Moreover, inhibition of phosphodiesterase type 5 (PDE5), a protein that selectively hydrolyzes cGMP, acts centrally within the SCN to enhance circadian responses adaptive to environmental changes in light, temperature, and nutrient availability. Likewise, PDE5 inhibitors (which are a cornerstone of PH treatment) directly affect homeostatic signaling of Per1 in a salutary manner that promotes organismal well-being (155).

Endothelin receptor antagonists.

PER1 signaling plays a role in positively modulating vascular health by regulating endothelial expression patterns. For example, investigators have reported a decrease in endothelin-1 (ET-1) expression in the lungs after heterozygous deletion of Per1 (197). This may in part explain the diurnal variation of ET-1 expression in even healthy adults (119, 198). It is unknown, however, what the role of endothelin receptor antagonists, another canonical PH medication drug class, plays in the circadian response of the endothelial tissue bed in health or disease.

Peroxisome proliferator-activated receptor.

The peroxisome proliferator-activated receptor (PPAR) family is intimately associated to circadian biology of the vascular endothelium, regulating the metabolic resilience of the pulmonary vascular bed and cardiomyocytes (199). In particular, PPAR-gamma (PPARγ) controls circadian variation in systemic blood pressure via direct regulation of BMAL1, whereby PPARγ-agonism upregulates Bmal1 mRNA expression and subsequent promotion of vascular health and recovery in response to injury (164). There is a fine balance, however, as central disruption of the light-dark cycle leads to aberrant neovascularization in the retina of susceptible mice via upregulation of PPARγ coactivator-1α (PGC-1α) (200). Although studied for some time, only recently has it been discovered that PPARγ-agonists prevent right heart failure in models of PH (201), although the rationale for the finding—through promotion of mitochondrial health—has been described (202, 203). Given the known safety profile of currently available PPARγ-agonist pioglitazone and the application of the drug class for decades to those patients with metabolic disease, consideration of circadian biology in application to PH treatment thus appears reasonable.

Resveratrol/Sirtuins.

Resveratrol is an activator of the Sirtuin system (Sirtuin 1) which is a family of deacetylase enzymes associated with metabolic health and robust natural aging. The drug has shown efficacy in the prevention and treatment of hypoxia-mediated and monocrotaline-induced PH through a slew of gross anti-inflammatory measures (204–206). Sirtuin 1 is likewise known to regulate the core clock through acetylation, facilitating expression of BMAL1/Per2 (207, 208). Related to lung circadian biology, Sirtuin 1 activation preserves BMAL1, leading to prevention of tobacco-mediated emphysematous changes in a murine model of chronic obstructive pulmonary disease (COPD) (209). However, there is additional evidence to support that the anti-inflammatory effects are secondary to lung epithelial cell expression of BMAL1 and not vascular cells (83).

mTOR inhibitors.

Related to cellular metabolism, as described above (see Peroxisome proliferator-activated receptor) mTOR is known to regulate central and peripheral clock function (158) serving to connect hypoxia and circadian-linked biology (210). Specifically, there is an observed increase in BMAL1 expression in a mTOR-activated state (211), with an expected increase in circadian regulatory protein Per2 (212). The latter acts primarily to suppress mTOR signaling. These findings can thus be interpreted through a lens of the large role mTOR plays in PH pathophysiology. Interestingly, pharmacologic inhibition of mTOR results in protection against elevated pulmonary vascular resistance and decreased right ventricular remodeling (89, 213). To date, low doses of mTOR inhibitor for this purpose have been demonstrated to be safe and potentially efficacious (214), with further trial results pending (ClinicalTrials.gov, NCT02587325). Therefore, considering the effects of circadian biology (as mentioned in the chronotherapy discussion below) could be therapeutically beneficial in these patients.

Drugging the Clock

Retinoic acid/retinoid.

There is a large and varied number of drugs targeting multiple specific ROR subtypes (128) that affect clock physiology. In particular, retinoids (ROR, RXR, and RAR) are highly expressed by human pulmonary artery smooth muscle cells. One study demonstrated that, in samples from patients with PAH there were quantitatively less circulating retinoid levels and decreased retinoid activity, leading to impaired proliferative signaling (168, 215). It is, therefore, interesting to speculate that retinoic acid signaling could serve to enhance the core clock regulation, resulting in protective effects against development of PH.

Melatonin.

Melatonin supplementation has been primarily examined in the context of re-entrainment of circadian homeostatic rhythmicity related to insomnia, neurodegenerative disorders (216), and cardiovascular disease (217). The mechanism in most cases focuses on the ability of melatonin to synchronize neuronal firing centrally in the SCN. The small number of studies examining the role of the natural hormone in PH describe a more peripheral effect of supplementation, leading to decreased inflammasome signaling and a decreased inflammatory milieu in the circulation. This is thought to be due mainly to melatonin’s antioxidant effects (218) with demonstrated efficacy in lowering pulmonary pressures in both the monocrotaline (186) and chronic hypoxia (219) model of disease.

Training the Clock

Sleep hygiene.

It is telling that the third most common symptom interfering with patient quality of life in PAH—only behind fatigue and dyspnea—is sleep difficulty (220), with degree of sleep disturbance positively correlating with severity of disease (221). However, it should be noted that this may also be due to signs/symptoms of heart failure, such as leg pain due to edema or orthopnea. Although there are no data that sleep fragmentation alone contributes to PH, it is considered a known contributor to pulmonary vascular remodeling secondary to OSA, which involves both endothelial dysfunction and structural vessel changes (222). Patients with sleep fragmentation due to OSA also have an increase in circulating MDSC with ROR expression, although these findings appear to be more related to intermittent hypoxia than disruption of sleep patterns (223).

Still, sleep influences healthy transcriptome profiles in both animals as well as humans (224), surprisingly through both the expected central regulatory machinery and through changes in the peripheral tissues (225). These effects are known to acutely alter the health of even young adults, with sleep fragmentation resulting in gross changes in body temperature, systemic blood pressure, and sympathetic tone (226). Given the broad health maintenance recommendations for improved sleep hygiene in the general population (227–229), there appears to be very little down side to at least emphasizing the role of restorative sleep in patients with PH.

Caloric Restriction and Time-Restricted Feeding

Restricted caloric intake, and as likely the timing of caloric intake, is a firm regulator of circadian metabolism. Proper metabolic functioning is an essential component of overall health and can heavily affect disease outcome (179). However, initial studies on CR have been called into question based on the observation that by merely decreasing caloric intake, both mice and humans will naturally sequester feeding around the time of food provision. This assertion is supported by later literature demonstrating a clear role for time restriction in nutrient intake in global health and resilience (230, 231), likely mediated by BMAL1 signaling (232). Coinciding with the recent scientific and lay-press piqued interest in the health benefits of time-restricted feeding and intermittent fasting (233), a small but well-designed study has recently shown that restricting caloric intake by 35% in the rat monocrotaline model of pulmonary vascular disease can significantly improve pulmonary vasorelaxation (234). Although more work needs to be done to fully elucidate this relationship, there is currently great interest in the study of macronutrient intake and metabolic changes associated with all forms of PH (235, 236).

CONCLUSIONS

“A straight line is not the shortest distance between two points.”

—Madeleine L’Engle, a Wrinkle in Time

The distinction between time as a quantitative measure compared to a qualitative perception (as in, now is the “right time” to act) is well distinguished by the ancient Greek use of two terms for the concept: Kronos (the objective form) and Kairos (the subjective). When we are healthy, we tend to hardly note the passage of chronological time, often remaining blissfully unaware of our well-functioning body and mind. It is only when our rhythms are disturbed in one way or another that we begin to note the passage of time in unpleasant milestones: pain, anxiety, or, in the case of lung disease, breathlessness. To date, there has been much evidence suggesting a major role for circadian biology in the progression of lung disease. However, investigation into potential pathways involved in both circadian rhythms and pulmonary vascular disease remains largely unexplored. It is our belief that this is a missed opportunity given that circadian biology offers a common thread to many rich areas of study within the realm of pulmonary vascular disease, including mitochondrial metabolism, endothelial and smooth muscle cell proliferation in response to hypoxia, and the role of innate/adaptive immunity in disease progression. To this end, we highlight several outstanding challenges and propose potential experimental approaches to meet these prompts within the greater PH community:

1) Circadian biomarkers: there are few surrogate clinical markers for clock-health, aside from melatonin, which is primarily useful in the assessment of SCN/central clock function. A circulating, exhaled, or composite physiologic biomarker that reflects peripheral tissue circadian disruption is necessary to gain better insight into defining not only chronotype, but studying responses to described therapeutics.

2) “Chicken or egg?”: not unique to pulmonary circadian clock is the difficulty in teasing out the causal relationship of circadian biology to disease; does interruption of clock homeostasis contribute to pulmonary vascular disease, or does pulmonary vascular disease lead to clock disruption? Could both, either/or, of these phenomena be true? Only careful studies examining individual core circadian molecular clock components, such as those outlined above, can help to shed light on these relationships.

3) Environmental exposures: given the known influence of tobacco smoke on parenchymal lung cell rhythms, it stands to reason that the pulmonary vasculature would likewise have a tissue-level specific change in clock output in response to inhalant exposure, temperature, hypoxia, hypercapnia, etc. Regarding the latter, there is much work to be done in determining the role of chronic or intermittent hypoxia in clock changes, especially concerning infiltrating pulmonary immune cells where circadian transcriptomic changes have recently been shown to be very similar to those from patients with PAH (237).

4) Sleep in PH: the overall influence of sleep disruption in the progression of pulmonary vascular disease is virtually unknown, despite the ubiquitous nature of sleep as described earlier in various types of systemic vasculopathies.

Finally, and most importantly, by viewing disease through a circadian lens, there remains a chance to not only improve performance of currently approved drugs, but also discover new potentially disease-modifying therapies for the treatment of patients with PH. Such a new “wrinkle” could therefore feasibly play a key role in the future of symptom management, and beyond.

GRANTS

This work was supported by the Bayer Pulmonary Hypertension Accelerated Bayer (PHAB) Award; National Institutes of Health (NIH) Grants R01 HL142776, R01 HL142887 (to A.J.B.), R01 DK109570 (to M.L.G.), and R01 NS054794; National Science Foundation (NSF) Grants IOS 1656647 (to A.C.L.), and R01 HL153042 and U01 AG055137 (to K.A.E.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.J.B., A.N., C.A.W., and A.C.L. prepared figures; A.J.B., E.E., A.N., C.A.W., M.L.G., A.C.L., and K.A.E. drafted manuscript; A.J.B., E.E., A.N., M.L.G., A.C.L., and K.A.E. edited and revised manuscript; A.J.B., E.E., A.N., M.L.G., A.C.L., and K.A.E. approved final version of manuscript.