The balance of nitric oxide (⅐NO) and superoxide anion (O 2 . ) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ⅐NO. As eNOS is capable of generating both ⅐NO and O 2 . , we hypothesized that Hsp90 might also mediate eNOS-dependent O 2 . production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 g/ml; 17.8 M) and then stimulated with the calcium ionophore, A23187 (5 M). GA significantly decreased A23187-stimulated eNOS-dependent nitrite production (p < 0.001, n ‫؍‬ 4) and significantly increased A23187-stimulated eNOS-dependent O 2 . production (p < 0.001, n ‫؍‬ 8). A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ⅐NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O 2 . production.Nitric oxide (⅐NO) and superoxide anion (O 2 . ) play opposing roles in vascular biology. Nitric oxide generation is increased greatly when Hsp90 associates with eNOS 1 in endothelial cell cultures (1, 2). A decrease in the amount of Hsp90 co-precipitating with eNOS is associated with a decrease in ⅐NO production by pulmonary artery endothelial cells exposed to prolonged periods of hypoxia (3). Geldanamycin (GA) is an ansamycin antibiotic that binds to the ATP binding site of Hsp90, thereby inhibiting the ATP/ADP cycle required for the interaction with client proteins such as eNOS (2-4). GA has been used to demonstrate that ⅐NO production in mesentary arteries and rat aortas depends on Hsp90 activity, implying that factors adversely affecting this interaction between Hsp90 and eNOS may be one of the mechanisms for portal hypertension and increased vascular tone (2, 4). Taken together, these reports indicate that Hsp90 is critical for eNOS generation of ⅐NO.Emerging evidence suggests that under pathological conditions eNOS may also generate O 2. (5-

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...

This study determined alterations to nitric oxide (NO)-dependent dilation of skeletal muscle arterioles from obese (OZR) versus lean Zucker rats (LZR). In situ cremaster muscle arterioles from both groups were viewed via television microscopy, and vessel dilation was measured with a video micrometer. Arteriolar dilation to acetylcholine and sodium nitroprusside was reduced in OZR versus LZR, although dilation to aprikalim was unaltered. NO-dependent flow-induced arteriolar dilation (via parallel microvessel occlusion) was attenuated in OZR, impairing arteriolar ability to regulate wall shear rate. Vascular superoxide levels, as assessed by dihydroethidine fluorescence, were elevated in OZR versus LZR. Treatment of cremaster muscles of OZR with the superoxide scavengers polyethylene glycol-superoxide dismutase and catalase improved arteriolar dilation to acetylcholine and sodium nitroprusside and restored flow-induced dilation and microvascular ability to regulate wall shear rate. These results suggest that NO-dependent dilation of skeletal muscle microvessels in OZR is impaired due to increased levels of superoxide. Taken together, these data suggest that the development of diabetes and hypertension in OZR may be associated with an impaired skeletal muscle perfusion via an elevated vascular oxidant stress.

Approximately 70% of women with polycystic ovary syndrome (PCOS) have intrinsic insulin resistance (IR) above and beyond that associated with body mass, including dysfunctional glucose metabolism in adipose tissue (AT). In AT, analysis of the IRS/PI3-K/AKT pathway signaling components identified only GLUT4 expression to be significantly lower in PCOS patients and in control subjects with IR. We examined the role of miRNAs, particularly in the regulation of GLUT4, the insulin-sensitive glucose transporter, in the AT of PCOS and matched control subjects. PCOS AT was determined to have a differentially expressed miRNA profile, including upregulated miR-93, -133, and -223. GLUT4 is a highly predicted target for miR-93, while miR-133 and miR-223 have been demonstrated to regulate GLUT4 expression in cardiomyocytes. Expression of miR-93 revealed a strong correlation between the homeostasis model assessment of IR in vivo values and GLUT4 and miR-93 but not miR-133 and -223 expression in human AT. Overexpression of miR-93 resulted in downregulation of GLUT4 gene expression in adipocytes through direct targeting of the GLUT4 3′UTR, while inhibition of miR-93 activity led to increased GLUT4 expression. These results point to a novel mechanism for regulating insulin-stimulated glucose uptake via miR-93 and demonstrate upregulated miR-93 expression in all PCOS, and in non-PCOS women with IR, possibly accounting for the IR of the syndrome. In contrast, miR-133 and miR-223 may have a different, although yet to be defined, role in the IR of PCOS.