Ciprian B. Anea

Vascular Disease in Mice With a Dysfunctional Circadian Clock

Background— Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. A profound pattern exists in the time of day at which the death occurs; it is in the morning, when the endothelium is most vulnerable and blood pressure surges, that stroke and heart attack most frequently happen. Although the molecular components of circadian rhythms rhythmically oscillate in blood vessels, evidence of a direct function for the “circadian clock” in the progression to vascular disease is lacking. Methods and Results— In the present study, we found increased pathological remodeling and vascular injury in mice with aberrant circadian rhythms, Bmal1-knockout and Clock mutant. In addition, naive aortas from Bmal1-knockout and Clock mutant mice exhibit endothelial dysfunction. Akt and subsequent nitric oxide signaling, a pathway critical to vascular function, was significantly attenuated in arteries from Bmal1-knockout mice. Conclusions— Our data reveal a new role for the circadian clock during chronic vascular responses that may be of significance in the progression of vascular disease.

Increased Superoxide and Endothelial NO Synthase Uncoupling in Blood Vessels of Bmal1-Knockout Mice

Rationale: Disruption of the circadian clock in mice produces vascular dysfunction as evidenced by impairments in endothelium-dependent signaling, vasomotion, and blood vessel remodeling. Although the altered function of endothelial NO synthase and the overproduction of reactive oxygen species are central to dysfunction of the endothelium, to date, the impact of the circadian clock on endothelial NO synthase coupling and vascular reactive oxygen species production is not known. Objective: The goals of the present study were to determine whether deletion of a critical component of the circadian clock, Bmal1, can influence endothelial NO synthase coupling and reactive oxygen species levels in arteries from Bmal1-knockout (KO) mice. Methods and Results: Endothelial function was reduced in aortae from Bmal1-KO mice and improved by scavenging reactive oxygen species with polyethylene glycol-superoxide dismutase and nonselectively inhibiting cyclooxygenase isoforms with indomethacin. Aortae from Bmal1-KO mice exhibited enhanced superoxide levels as determined by electron paramagnetic resonance spectroscopy and dihydroethidium fluorescence, an elevation that was abrogated by administration of nitro-L-arginine methyl ester. High-performance liquid chromatography analysis revealed a reduction in tetrahydrobiopterin and an increase in dihydrobiopterin levels in the lung and aorta of Bmal1-KO mice, whereas supplementation with tetrahydrobiopterin improved endothelial function in the circadian clock KO mice. Furthermore, levels of tetrahydrobiopterin, dihydrobiopterin, and the key enzymes that regulate biopterin bioavailability, GTP cyclohydrolase and dihydrofolate reductase exhibited a circadian expression pattern. Conclusions: Having an established influence in the metabolic control of glucose and lipids, herein, we describe a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of endothelial NO synthase, production of superoxide, and maintenance of 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.

Matrix Metalloproteinase 2 and 9 Dysfunction Underlie Vascular Stiffness in Circadian Clock Mutant Mice

Objective—To determine if elasticity in blood vessels is compromised in circadian clock–mutant mice (Bmal1-knockout [KO] and Per-triple KO) and if matrix metalloproteinases (MMPs) might confer these changes in compliance. Methods and Results—High-resolution ultrasonography in vivo revealed impaired remodeling and increased pulse-wave velocity in the arteries of Bmal1-KO and Per-triple KO mice. In addition, compliance of remodeled arteries and naïve pressurized arterioles ex vivo from Bmal1-KO and Per-triple KO mice was reduced, consistent with stiffening of the vascular bed. The observed vascular stiffness was coincident with dysregulation of MMP-2 and MMP-9 in Bmal1-KO mice. Furthermore, inhibition of MMPs improved indexes of pathological remodeling in wild-type mice, but the effect was abolished in Bmal1-KO mice. Conclusion—Circadian clock dysfunction contributes to hardening of arteries, which may involve impaired control of the extracellular matrix composition.

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.

Pulmonary Platelet Thrombi and Vascular Pathology in Acute Chest Syndrome in Patients with Sickle Cell Disease

A growing body of evidence suggests a role for platelets in sickle cell disease (SCD). Despite the proinflammatory, occlusive nature of platelets, a role for platelets in acute chest syndrome (ACS), however, remains understudied. To provide evidence and potentially describe contributory factors for a putative link between ACS and platelets, we performed an autopsy study of 20 SCD cases—10 of whom died from ACS and 10 whose deaths were not ACS‐related. Pulmonary histopathology and case history were collected. We discovered that disseminated pulmonary platelet thrombi were present in 3 out of 10 of cases with ACS, but none of the matched cases without ACS. Those cases with detected thrombi were associated with significant deposition of endothelial vWF and detection of large vWF aggregates adhered to endothelium. Potential clinical risk factors were younger age and higher platelet count at presentation. However, we also noted a sharp and significant decline in platelet count prior to death in each case with platelet thrombi in the lungs. In this study, neither hydroxyurea use nor perimortem transfusion was associated with platelet thrombi. Surprisingly, in all cases, there was profound pulmonary artery remodeling with both thrombotic and proliferative pulmonary plexiform lesions. The severity of remodeling was not associated with a severe history of ACS, or hydroxyurea use, but was inversely correlated with age. We thus provide evidence of undocumented presence of platelet thrombi in cases of fatal ACS and describe clinical correlates. We also provide novel correlates of pulmonary remodeling in SCD. Am. J. Hematol. 91:173–178, 2016.

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.

Immunohistochemistry of the circadian clock in mouse and human vascular tissues

Aim The circadian clock is a molecular network that controls the body physiological rhythms. In blood vessels, the circadian clock components modulate vascular remodeling, blood pressure, and signaling. The goal in this study was to determine the pattern of expression of circadian clock proteins in the endothelium, smooth muscle, and adventitia of the vasculature of human and mouse tissues. Methods Immunohistochemistry was performed in frozen sections of mouse aorta, common carotid artery, femoral artery, lung, and heart at 12 AM and 12 PM for Bmal1, Clock, Npas2, Per and other clock components. Studies of expression were also assessed in human saphenous vein both by immunoblotting and immunohistochemistry. Results In this study, we identified the expression of Bmal1, Clock, Npas, Per1, Cry1, and accessory clock components by immunohistochemical staining in the endothelium, smooth muscle and adventitia of the mouse vasculature with differing temporal and cellular profiles depending on vasculature and tissue analyzed. The human saphenous vein also exhibited expression of clock genes that exhibited an oscillatory pattern in Bmal1 and Cry by immunoblotting. Conclusion These studies show that circadian clock components display differences in expression and localization throughout the cardiovascular system, which may confer nuances of circadian clock signaling in a cell-specific manner.