Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NF B-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.Endothelial cells are constantly exposed to shear stress (a dragging force generated by blood flow), which controls cellular structure and function such as regulation of vascular tone and diameter, vessel wall remodeling, hemostasis, and inflammatory responses (1). The importance of various types of shear stress is highlighted by the focal development of atherosclerosis (2). Atherosclerosis preferentially occurs in the arterial regions exposed to unstable shear stress conditions in branched or curved arteries, whereas straight arteries exposed to unidirectional laminar shear (LS) 1 are relatively lesion-free (1-3). Atherosclerosis is now known as an inflammatory disease caused by endothelial dysfunction (3, 4). One of the first visible markers of endothelial dysfunction in the lesion-prone areas is upregulation of inflammatory adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 (3-6). These endothelial adhesion molecules play essential roles in adhesion and recruitment of monocytes to the subendothelial layer (3, 4).How do unstable shear conditions such as low and oscillating shear stress (OS) cause inflammation in those lesion-prone areas, whereas LS exerts athero-protective effects? The opposite effects of LS and OS may be determined by differential expression of genes and proteins, ultimately inducing anti-and pro-inflammatory and atherogenic responses. Recently, several studies (7-10) have begun to address the initial question to determine the expression profiles of mechanosensitive genes. However, the functional importance of those genes has not been clearly established.Here, we report identification of a mechanosensitive gene, BMP4, by DNA microarray analyses and subsequent verification by a variety of additional approac...

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O 2 ؊ from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47 phox؊/؊ (MAE-p47 ؊/؊ ) mice, which lack a component of NAD(P)H oxidase. O 2 ؊ production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (؎ 5 dynes/cm 2 ) increased O 2 ؊ (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm 2 , 18 h) significantly decreased both monocyte adhesion and O 2 ؊ compared with static conditions. In contrast, neither LS nor OS were able to induce O 2 ؊ production and monocyte adhesion to MAE-p47 ؊/؊ . Treating MAE-C57 with a cellpermeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47 phox in MAE-p47 ؊/؊ restored OS-induced O 2 ؊ production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O 2 ؊ and/or its derivatives produced from p47 phox -dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.Fluid shear stress, the frictional force generated by blood flow over the vascular endothelium, is a major factor in atherogenesis. The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis in hemodynamically defined regions. For example, regions of branched and curved arteries experience disturbed blood flow patterns, including oscillatory shear stress (OS), 1 typically ranging Ϯ 5 dynes/cm 2 (Ϯ indicates changes in flow directions) (1, 2). These disturbed shear regions correspond to "lesion-prone" areas that develop early forms of atherosclerotic lesions (1-3). In contrast, relatively straight arteries, which are exposed to steady uni-directional laminar shear stress (typically ranging from 5-25 dynes/cm 2 ), are usually protected from early atherosclerotic plaque development (1, 2). The mechanisms by which laminar shear (LS) acts as atheroprotective force whereas OS initiates or contributes to atherogenesis have been the subject of intense investigation by many researchers.The vascular endothelium is in direct contact with blood flow and acts as a mechanotransducer by sensing and transducing the changes in local mechanical forces into cellular signals. Endothelial function, shape, physiology, and pathophysiology are greatly regulated by the types (uni-directional laminar or disturbed flow conditions) and magnitudes (high or...

Background Due to the devastating late effects associated with cranial irradiation in young children with CNS tumors, treatment for these patients has evolved to include the use of intensive chemotherapy to either avoid or postpone irradiation. While survival outcomes have improved, late effects data in survivors treated on such regimens are needed. Objective This multi-institutional study comprehensively describes late effects in survivors treated on the Head Start I/II protocols. Methods Survivors of CNS tumors treated on Head Start I/II protocols were enrolled. Late effects data were collected using a validated parent-report questionnaire. Social, emotional, and behavioral functioning and quality of life were assessed using parent-report on the BASC-2 and CHQ-PF50 questionnaires. Results Twenty one survivors (medulloblastoma=13, sPNET=4, ATRT=1, ependymoma=3) were enrolled. Ten (48%) were irradiation-free. Late effects (frequency; median time of onset since diagnosis) included ≥ grade III hearing loss (67%; 3.9 years), vision (67%; 4.1 years), hypothyroidism (33%; 4 years), growth hormone (GH) deficiency (48%; 4.7 years) and dental (52%; 7.1 years) and no cases of secondary leukemia. Irradiation-free (versus irradiated) survivors reported low rates of hypothyroidism (0/10 vs 7/11; p=0.004) and GH deficiency (2/10 vs 8/11; p=0.03). The BASC-2 and CHQPF-50 mean composite scores were within average ranges relative to healthy comparison norms. Neither age at diagnosis nor irradiation were associated with these scores. Conclusions Irradiation-free Head Start survivors have lower risk of hypothyroidism and GH deficiency. Secondary leukemias are not reported. With extended follow up, survivors demonstrate quality of life, social, emotional, and behavioral functioning within average ranges.