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...

Expression profiling has identified metastasis-associated microRNAs (miRNA) but technical limitations hinder the discovery of metastasis-suppressing miRNAs. In this study, we sought metastasis-suppressing miRNAs by functional screening. Individual miRNAs were lentivirally introduced into metastatic MDA-MB-231 breast cancer cells and analyzed for effects on cell migration, a critical step in cancer metastasis. Among 486 miRNAs screened, 14 were identified that included all of the members of the miRNA-196 family (miR-196a1, miR-196a2, and miR-196b). Enforced expression of miR-196a1/2 or miR-196b abrogated in vitro invasion and in vivo spontaneous metastasis of breast cancer cells, indicating that members of the miR-196 family are potent metastasis suppressors. We found that miR-196 inhibited the expression of transcription factor HOXC8. Functional linkage was implied by small interfering RNA-mediated knockdown of HOXC8, which suppressed cell migration and metastasis, and by ectopic expression of HOXC8, which prevented the effects of miR-196 on cell migration and metastasis. Unlike other metastasis-associated miRNAs that have been described, the expressions of miR-196 were not correlated with breast cancer cell migration or the metastatic status of clinical breast tumor specimens. Instead, we detected an excellent correlation between the ratio of miR-196 to HOXC8 messages and the migratory behavior of breast cancer cell lines as well as the metastatic status of clinical samples. Our findings identify miRNA-196s as potent metastasis suppressors and reveal that the ratio of miR-196s to HOXC8 mRNA might be an indicator of the metastatic capability of breast tumors. Cancer Res; 70(20); 7894-904.

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of ␣ 2 -adrenergic receptors (␣ 2 -ARs) by GGA3 (Golgi-localized, ␥-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized ␣ 2B -AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated ␣ 2B -AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with ␣ 2B -AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, ␣ 2A -AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

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.