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. Key Words: circadian rhythm Ⅲ endothelium Ⅲ remodeling Ⅲ thrombus Ⅲ vasculature T he cardiovascular system behaves rhythmically over the course of a day, 1-3 coordinating tissue perfusion in accordance with oscillating metabolic and functional demands. These oscillations, which occur in blood vessels as variations in contractility and blood pressure, follow a distinctive temporal pattern-a circadian rhythm. Aberrations to circadian rhythm meet with pathological consequences. Shift work provokes a 40% increase in the risk of cardiovascular disease, 4 and disturbance of daily blood pressure rhythms elevates the incidence of vascular disease. 5,6 In addition, the onset of acute vascular events such as myocardial infarction 7 and stroke 8 also exhibits circadian variation. However, direct evidence to implicate the molecular components of circadian rhythms in the chronic progression of disease is lacking. Conclusions-Our Editorial p 1463 Clinical Perspective p 1517The molecular components that generate circadian rhythms-the circadian/biological clock-constitute a unique collaboration of genes and proteins that govern by virtue of transcriptional, translational, and posttranslational mechanisms. The transcriptional driving force is composed of the basic helix-loop-helix transcription factors Bmal1 and Clock (or Npas2). Bmal1/Clock or Bmal1/ Npas2 heterodimerize to transactivate Period (Per) and Cryptochrome (Cry), the braking force of the loop, which then restrain Bmal1/Clock/Npas2 and, consequently, their own transcription. Further modification of the core complex, including phosphorylation and degradation, refines timing to the daily cycle. 9 Indeed, vascular cells 10,11 contain all necessary components of this unique molecular metronome. Although recent data have implicated the circadian clock in aspects of acute vascular function, [12][13][14] no data directly implicate Bmal1 or Clock in the chronic process of vascular disease, and even less is known on the downstream mechanisms involved. Methods AnimalsAll animal studies were performed according to protocols app...

Endothelin-1 (ET-1) is implicated in the development of endothelial dysfunction through the generation of reactive oxygen species by NADPH oxidase activation. Interleukin-10 (IL-10) is an antiinflammatory cytokine that stimulates nitric oxide production, decreases superoxide production, and restores endothelial integrity after vascular injury. In this study, we tested whether IL-10 attenuates ET-1-induced endothelial dysfunction by improving acetylcholine (ACh)-induced relaxation of cultured murine aortic rings. Aortic rings (2 mm long) of C57BL/6 mice were incubated in 2 mL DMEM containing 120 U/mL penicillin and 120 µg/mL streptomycin in the presence of one of 4 treatments: vehicle (deionized water), ET-1 (100 nmol/L), recombinant mouse IL-10 (300 ng/ mL), or a combination of both ET-1 and IL-10. After incubation at 37 °C for either 1 or 6 h (shortterm exposure) or 22 h (overnight exposure), rings were mounted in a wire myograph and stretched to a passive force of 5 mN. Endothelium-dependent vasorelaxation was assessed by constructing cumulative concentration-response curves to ACh (0.001-10 µmol/L) during 10 µmol/L phenylephrine (PE)-induced contraction. Short-term exposure of ET-1 did not result in an impairment of ACh-induced relaxation. Overnight exposure of aortic rings to ET-1 resulted in a statistically significant endothelial dysfunction characterized by a reduced maximal relaxation response to ACh compared with that of untreated rings (E max 57% ± 3% versus 82% ± 4%). IL-10 treatment restored ACh-induced relaxation (E max 77% ± 3%). Western blotting showed decreased eNOS expression in response to ET-1, whereas vessels treated with a combination of ET-1 and IL-10 showed increased expression of eNOS. Immunohistochemical analysis showed decreased eNOS expression in ET-1-treated vessels compared with those treated with both ET-1 and IL-10. We conclude that, in murine aorta, the antiinflammatory cytokine IL-10 prevents impairment in endothelium-dependent 1 This article is one of a selection of papers published in the special issue (part 2 of 2) on Forefronts in Endothelin.

Mutation of the leptin receptor in mice (db/db) causes striking obesity and insulin resistance which may negatively impact the vasculature. Surprisingly, previous studies have suggested that these obese mice are protected from vascular injury. However, denudation of the endothelium used to provoke injury in these studies may mask a protective role for leptin within the vascular endothelium. To address this possibility, we implemented a non‐denuding model of flow‐induced vascular remodeling. Endothelial integrity in remodeled arteries was preserved as assessed by PECAM immunohistochemistry in both control (lean) mice and db/db (obese) mice. Arteries of lean mice exhibited typical inward vascular remodeling as expected, but remodeled vessels of obese mice exhibited robust intimal hyperplasia in response to the remodeling stimulus. To assess the impact of glycemia on the vascular remodeling response, we generated mice that were obese but normoglycemic (obese‐NG). This was accomplished by intercrossing the obese mice that were deficient in the leptin receptor to mice deficient in PTP1B, an insulin receptor phosphatase, which when disrupted in mice, causes insulin hypersensitivity. Though blood glucose levels were elevated in obese mice, blood glucose was restored to normal in the doubly transgenic obese‐NG mice, as revealed by levels of glycosylated hemoglobin (obese, 9.48%±0.44; obese‐NG, 6.84%±0.44; lean, 4.98%±0.14). Furthermore, remodeled arteries from the obese‐NG mice were protected from the neointimal formation that occurred in the obese mice. These data suggest that the leptin pathway restrains PTP1B signaling to improve glycemia and retard pathological remodeling.

Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. There is a profound pattern 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. Though the biological clock rhythmically oscillates in blood vessels, evidence of a direct function in the vasculature for Bmal1, an essential clock component, is lacking. We have found that chronic flow interruption in arteries of mice with targeted disruption of Bmal1 gene (Bmal1-KO) causes pathological vascular remodeling which worsens to thrombosis with ageing. In addition, naïve aortic arteries of Bmal1-KO mice exhibit endothelial dysfunction, which is improved by relief of oxidant stress. Akt signalling, a pathway critical to vascular function, was significantly attenuated in arteries of Bmal1-KO mice. Co-transfection of cultured cells with the transcription factors Bmal-Npas2 or Bmal1-Clock induced a robust activation of the Akt promoter, providing evidence of a direct link between the circadian clock and Akt pathways. Our data reveals a new role for the biological clock in the control of endothelial signalling and vascular remodeling which may be of significant impact in the progression of vascular disease.