Chronic mistimed feeding results in renal fibrosis and disrupted circadian blood pressure rhythms.

Circadian disruption is a disturbance in biological timing which can occur within or between different organizational levels, ranging from molecular rhythms within specific cells to misalignment of behavioral and environmental cycles. Previous work from our group showed that less than one week of food restriction to the light (inactive) period is sufficient to invert diurnal blood pressure rhythms in mice. However, kidney excretory rhythms and functions remained aligned with the light-dark cycle. Shift workers have increased risk of cardiovascular disease that may different between sexes and often have irregular mealtimes making the possibility of mistimed feeding as a potential contributor to the development of kidney disease. Thus, we hypothesized that chronic mistimed food intake would result in adverse cardiorenal effects with sex-differences in severity. Here we show that chronic circadian disruption via mistimed feeding results in renal fibrosis and aortic stiffness in a sex-dependent manner. Our results indicate the importance of meal timing for maintenance of blood pressure rhythms and kidney function, particularly in males. Our results also demonstrate females are better able to acclimate to circadian-related behavioral change.

Liver circadian clock disruption alters perivascular adipose tissue gene expression and aortic function in mice.

The liver plays a central role that influences cardiovascular disease outcomes through regulation of glucose and lipid metabolism. It is recognized that the local liver molecular clock regulates some liver-derived metabolites. However, it is unknown whether the liver clock may impact cardiovascular function. Perivascular adipose tissue (PVAT) is a specialized type of adipose tissue surrounding blood vessels. Importantly, cross talk between the endothelium and PVAT via vasoactive factors is critical for vascular function. Therefore, we designed studies to test the hypothesis that cardiovascular function, including PVAT function, is impaired in mice with liver-specific circadian clock disruption. Bmal1 is a core circadian clock gene, thus studies were undertaken in male hepatocyte-specific Bmal1 knockout (HBK) mice and littermate controls (i.e., flox mice). HBK mice showed significantly elevated plasma levels of ß-hydroxybutyrate, nonesterified fatty acids/free fatty acids, triglycerides, and insulin-like growth factor 1 compared with flox mice. Thoracic aorta PVAT in HBK mice had increased mRNA expression of several key regulatory and metabolic genes, Ppargc1a, Pparg, Adipoq, Lpl, and Ucp1, suggesting altered PVAT energy metabolism and thermogenesis. Sensitivity to acetylcholine-induced vasorelaxation was significantly decreased in the aortae of HBK mice with PVAT attached compared with aortae of HBK mice with PVAT removed, however, aortic vasorelaxation in flox mice showed no differences with or without attached PVAT. HBK mice had a significantly lower systolic blood pressure during the inactive period of the day. These new findings establish a novel role of the liver circadian clock in regulating PVAT metabolic gene expression and PVAT-mediated aortic vascular function.

Differential Regulation of BMAL1, CLOCK, and Endothelial Signaling in the Aortic Arch and Ligated Common Carotid Artery.

The circadian clock is rhythmically expressed in blood vessels, but the interaction between the circadian clock and disturbed blood flow remains unclear. We examined the relationships between BMAL1 and CLOCK and 2 regulators of endothelial function, AKT1 and endothelial nitric oxide synthase (eNOS), in vascular regions of altered blood flow. We found that the aortic arch from WT mice exhibited reduced sensitivity to acetylcholine (Ach)-mediated relaxation relative to the thoracic aorta. In Clock-mutant (mut) mice the aorta exhibited a reduced sensitivity to Ach. In WT mice, the phosphorylated forms of eNOS and AKT were decreased in the aortic arch, while BMAL1 and CLOCK expression followed a similar pattern of reduction in the arch. In conditions of surgically induced flow reduction, phosphorylated-eNOS (serine 1177) increased, as did p-AKT in the ipsilateral left common carotid artery (LC) of WT mice. Similarly, BMAL1 and CLOCK exhibited increased expression after 5 days in the remodeled LC. eNOS expression was increased at 8 p.m. versus 8 a.m. in WT mice, and this pattern was abolished in mut and Bmal1-KO mice. These data suggest that the circadian clock may be a biomechanical and temporal sensor that acts to coordinate timing, flow dynamics, and endothelial function.

Tissue-intrinsic dysfunction of circadian clock confers transplant arteriosclerosis.

The suprachiasmatic nucleus of the brain is the circadian center, relaying rhythmic environmental and behavioral information to peripheral tissues to control circadian physiology. As such, central clock dysfunction can alter systemic homeostasis to consequently impair peripheral physiology in a manner that is secondary to circadian malfunction. To determine the impact of circadian clock function in organ transplantation and dissect the influence of intrinsic tissue clocks versus extrinsic clocks, we implemented a blood vessel grafting approach to surgically assemble a chimeric mouse that was part wild-type (WT) and part circadian clock mutant. Arterial isografts from donor WT mice that had been anastamosed to common carotid arteries of recipient WT mice (WT:WT) exhibited no pathology in this syngeneic transplant strategy. Similarly, when WT grafts were anastamosed to mice with disrupted circadian clocks, the structural features of the WT grafts immersed in the milieu of circadian malfunction were normal and absent of lesions, comparable to WT:WT grafts. In contrast, aortic grafts from Bmal1 knockout (KO) or Period-2,3 double-KO mice transplanted into littermate control WT mice developed robust arteriosclerotic disease. These lesions observed in donor grafts of Bmal1-KO were associated with up-regulation in T-cell receptors, macrophages, and infiltrating cells in the vascular grafts, but were independent of hemodynamics and B and T cell-mediated immunity. These data demonstrate the significance of intrinsic tissue clocks as an autonomous influence in experimental models of arteriosclerotic disease, which may have implications with regard to the influence of circadian clock function in organ transplantation.

Timed feeding aligns the adipocyte clock to optimize thermogenesis when eating a high-fat diet.

The timing of food intake is vital for metabolic health in obesity. A recent study in mice from Hepler et al. in Science shows the importance of the adipocyte circadian clock in metabolic health, highlighting the creatine pathway and thermogenesis with the alignment of the timing of high-fat feeding.

Timing of Food Intake Drives the Circadian Rhythm of Blood Pressure.

Timing of food intake has become a critical factor in determining overall cardiometabolic health. We hypothesized that timing of food intake entrains circadian rhythms of blood pressure (BP) and renal excretion in mice. Male C57BL/6J mice were fed ad libitum or reverse feeding (RF) where food was available at all times of day or only available during the 12-h lights-on period, respectively. Mice eating ad libitum had a significantly higher mean arterial pressure (MAP) during lights-off compared to lights-on (113 ± 2 mmHg vs 100 ± 2 mmHg, respectively; P < 0.0001); however, RF for 6 days inverted the diurnal rhythm of MAP (99 ± 3 vs 110 ± 3 mmHg, respectively; P < 0.0001). In contrast to MAP, diurnal rhythms of urine volume and sodium excretion remained intact after RF. Male Bmal1 knockout mice (Bmal1KO) underwent the same feeding protocol. As previously reported, Bmal1KO mice did not exhibit a diurnal MAP rhythm during ad libitum feeding (95 ± 1 mmHg vs 92 ± 3 mmHg, lights-off vs lights-on; P > 0.05); however, RF induced a diurnal rhythm of MAP (79 ± 3 mmHg vs 95 ± 2 mmHg, lights-off vs lights-on phase; P < 0.01). Transgenic PERIOD2::LUCIFERASE knock-in mice were used to assess the rhythm of the clock protein PERIOD2 in ex vivo tissue cultures. The timing of the PER2::LUC rhythm in the renal cortex and suprachiasmatic nucleus was not affected by RF; however, RF induced significant phase shifts in the liver, renal inner medulla, and adrenal gland. In conclusion, the timing of food intake controls BP rhythms in mice independent of Bmal1, urine volume, or sodium excretion.

Increased arterial pressure in mice with overexpression of the ADHD candidate gene calcyon in forebrain.

The link between blood pressure (BP) and cerebral function is well established. However, it is not clear whether a common mechanism could underlie the relationship between elevated BP and cognitive deficits. The expression of calcyon, a gene abundant in catecholaminergic and hypothalamic nuclei along with other forebrain regions, is increased in the brain of the spontaneously hypertensive rat (SHR) which is a widely accepted animal model of essential hypertension and attention deficit hyperactivity disorder (ADHD). Previous studies demonstrated that mice with up-regulation of calcyon in forebrain (CalOE) exhibit deficits in working memory. To date, there is no evidence directly connecting calcyon to BP regulation. Here, we investigated whether forebrain up-regulation of calcyon alters BP using radiotelemetry. We found that CalOE mice exhibited higher mean arterial pressure (MAP) compared to tTA controls. Plasma norepinephrine levels were significantly higher in CalOE mice compared to tTA controls. Silencing the transgene with doxycycline normalized BP in CalOE mice, whereas challenging the mice with 4% high salt diet for 12 days exacerbated the MAP differences between CalOE and tTA mice. High salt diet challenge also increased proteinuria and urinary thiobarbituric acid reactive substances (TBARs) in tTA and CalOE; and the increases were more prominent in CalOE mice. Taken together, our data suggest that upregulation of calcyon in forebrain could increase BP via alterations in noradrenergic transmission and increased oxidative stress during high salt challenge. Overall, this study reveals that calcyon could be a novel neural regulator of BP raising the possibility that it could play a role in the development of vascular abnormalities.

Genetic Dissection of the Impact of miR-33a and miR-33b during the Progression of Atherosclerosis.

As an important regulator of macrophage cholesterol efflux and HDL biogenesis, miR-33 is a promising target for treatment of atherosclerosis, and numerous studies demonstrate that inhibition of miR-33 increases HDL levels and reduces plaque burden. However, important questions remain about how miR-33 impacts atherogenesis, including whether this protection is primarily due to direct effects on plaque macrophages or regulation of lipid metabolism in the liver. We demonstrate that miR-33 deficiency in Ldlr-/- mice promotes obesity, insulin resistance, and hyperlipidemia but does not impact plaque development. We further assess how loss of miR-33 or addition of miR-33b in macrophages and other hematopoietic cells impact atherogenesis. Macrophage-specific loss of miR-33 decreases lipid accumulation and inflammation under hyperlipidemic conditions, leading to reduced plaque burden. Therefore, the pro-atherogenic effects observed in miR-33-deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.

microRNAs in lipoprotein metabolism and cardiometabolic disorders.

Circulating levels of low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) are two of the most important risk factors for the development of cardiovascular disease (CVD), the leading cause of death worldwide. Recently, miRNAs have emerged as critical regulators of cholesterol metabolism and promising therapeutic targets for the treatment of CVD. A great deal of work has established numerous miRNAs as important regulators of HDL metabolism. This includes miRNAs that target ABCA1, a critical factor for HDL biogenesis and reverse cholesterol transport (RCT), the process through which cells, including arterial macrophages, efflux cellular cholesterol for transport to and removal by the liver. The most well studied of these miRNAs, miR-33, has been demonstrated to target ABCA1, as well as numerous other genes involved in metabolic function and RCT, and therapeutic inhibition of miR-33 was found to increase HDL levels in mice and non-human primates. Moreover, numerous studies have demonstrated the beneficial effects of miR-33 inhibition or knockout on reducing atherosclerotic plaque burden. Even more recent work has identified miRNAs that regulate LDL cholesterol levels, including direct modulation of LDL uptake in the liver through targeting of the LDL receptor. Among these, inhibition of miR-128-1, miR-148a, or miR-185 was found to reduce plasma LDL levels, and inhibition of miR-185 was further demonstrated to reduce atherosclerotic plaque size in ApoE(-/-) mice. Due to their ability to target many different genes, miRNAs have the ability to mediate complex physiologic changes through simultaneous regulation of multiple interrelated pathways. Of particular importance for CVD, inhibition of miR-148a may prove an important therapeutic approach for combating dyslipidemia, as this has been demonstrated to both raise plasma HDL levels and lower LDL levels in mice by targeting both ABCA1 and LDLR, respectively. In this review we highlight recent advances in our understanding of how miRNAs regulate cholesterol metabolism and the development of atherosclerotic plaques and discuss the potential of anti-miRNA therapies for the treatment and prevention of CVD.

Low-Salt Diet and Circadian Dysfunction Synergize to Induce Angiotensin II-Dependent Hypertension in Mice.

Blood pressure exhibits a robust circadian rhythm in health. In hypertension, sleep apnea, and even shift work, this balanced rhythm is perturbed via elevations in night-time blood pressure, inflicting silent damage to the vasculature and body organs. Herein, we examined the influence of circadian dysfunction during experimental hypertension in mice. Using radiotelemetry to measure ambulatory blood pressure and activity, the effects of angiotensin II administration were studied in wild-type (WT) and period isoform knockout (KO) mice (Per2-KO, Per2, 3-KO, and Per1, 2, 3-KO/Per triple KO [TKO] mice). On a normal diet, administration of angiotensin II caused nondipping blood pressure and exacerbated vascular hypertrophy in the Period isoform KO mice relative to WT mice. To study the endogenous effects of angiotensin II stimulation, we then administered a low-salt diet to the mice, which does stimulate endogenous angiotensin II in addition to lowering blood pressure. A low-salt diet decreased blood pressure in wild-type mice. In contrast, Period isoform KO mice lost their circadian rhythm in blood pressure on a low-salt diet, because of an increase in resting blood pressure, which was restorable to rhythmicity by the angiotensin receptor blocker losartan. Chronic administration of low salt caused vascular hypertrophy in Period isoform KO mice, which also exhibited increased renin levels and altered angiotensin 1 receptor expression. These data suggest that circadian clock genes may act to inhibit or control renin/angiotensin signaling. Moreover, circadian disorders such as sleep apnea and shift work may alter the homeostatic responses to sodium restriction to potentially influence nocturnal hypertension.

Endotoxin Disrupts Circadian Rhythms in Macrophages via Reactive Oxygen Species.

The circadian clock is a transcriptional network that functions to regulate the expression of genes important in the anticipation of changes in cellular and organ function. Recent studies have revealed that the recognition of pathogens and subsequent initiation of inflammatory responses are strongly regulated by a macrophage-intrinsic circadian clock. We hypothesized that the circadian pattern of gene expression might be influenced by inflammatory stimuli and that loss of circadian function in immune cells can promote pro-inflammatory behavior. To investigate circadian rhythms in inflammatory cells, peritoneal macrophages were isolated from mPer2luciferase transgenic mice and circadian oscillations were studied in response to stimuli. Using Cosinor analysis, we found that LPS significantly altered the circadian period in peritoneal macrophages from mPer2luciferase mice while qPCR data suggested that the pattern of expression of the core circadian gene (Bmal1) was disrupted. Inhibition of TLR4 offered protection from the LPS-induced impairment in rhythm, suggesting a role for toll-like receptor signaling. To explore the mechanisms involved, we inhibited LPS-stimulated NO and superoxide. Inhibition of NO synthesis with L-NAME had no effect on circadian rhythms. In contrast, inhibition of superoxide with Tempol or PEG-SOD ameliorated the LPS-induced changes in circadian periodicity. In gain of function experiments, we found that overexpression of NOX5, a source of ROS, could significantly disrupt circadian function in a circadian reporter cell line (U2OS) whereas iNOS overexpression, a source of NO, was ineffective. To assess whether alteration of circadian rhythms influences macrophage function, peritoneal macrophages were isolated from Bmal1-KO and Per-TKO mice. Compared to WT macrophages, macrophages from circadian knockout mice exhibited altered balance between NO and ROS release, increased uptake of oxLDL and increased adhesion and migration. These results suggest that pro-inflammatory stimuli can disrupt circadian rhythms in macrophages and that impaired circadian rhythms may contribute to cardiovascular diseases by altering macrophage behavior.

Circadian clock control of Nox4 and reactive oxygen species in the vasculature.

Recent studies have shown that circadian clock disruption is associated with pathological remodeling in the arterial structure and vascular stiffness. Moreover, chronic circadian disruption is associated with dysfunction in endothelial responses and signaling. Reactive oxygen species have emerged as key regulators in vascular pathology. Previously, we have demonstrated that circadian clock dysfunction exacerbates superoxide production through eNOS uncoupling. To date, the impact of circadian clock mutation on vascular NADPH oxidase expression and function is not known. The goal in the current study was to determine if the circadian clock controls vascular Nox4 expression and hydrogen peroxide formation in arteries, particularly in endothelial and vascular smooth muscle cells. In aorta, there was an increase in hydrogen peroxide and Nox4 expression in mice with a dysfunctional circadian rhythm (Bmal1-KO mice). In addition, the Nox4 gene promoter is activated by the core circadian transcription factors. Lastly, in synchronized cultured human endothelial cells, Nox4 gene expression exhibited rhythmic oscillations. These data reveal that the circadian clock plays an important role in the control of Nox4 and disruption of the clock leads to subsequent production of reaction oxygen species.