Abstract-Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H 2 O 2 and O 2 Ϫ production in ECs. Next, we used ECs obtained from p47phox Ϫ/Ϫ mice (MAE-p47 Ϫ/Ϫ ), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47 Ϫ/Ϫ , but it was restored when the cells were transfected with p47 phox plasmid. Moreover, OS-induced O 2 Ϫ production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step. Key words: BMP4 Ⅲ oscillatory shear Ⅲ reactive oxygen species Ⅲ monocyte adhesion Ⅲ endothelial cells Ⅲ NADPH oxidase V ascular endothelial cells (ECs) are constantly exposed to fluid shear stress, the frictional force generated by blood flow over the vascular endothelium. 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, the regions of branched and curved arteries exposed to disturbed flow conditions including oscillatory shear stress (OS) correspond to "lesion-prone areas" that preferentially develop atherosclerosis. 1,2 In contrast, straight arteries exposed to steady, high levels of laminar shear stress (LS) are relatively well protected from atherosclerotic plaque development. 1,2 Atherosclerosis is an inflammatory disease preferentially occurring in lesion-prone areas. 2,3 The earliest measurable markers of atherogenesis include expression of inflammatory adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and subsequent monocyte adhesion and recruitment into the lesion-prone areas. 2,4,5 Additional critical atherogen...

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...

Hypertension caused by angiotensin II is dependent on vascular superoxide (O2*-) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O2*- and is activated by angiotensin II in vitro. However, its role in angiotensin II-induced hypertension in vivo is less clear. In the present studies, we used mice deficient in p47(phox), a cytosolic subunit of the NADPH oxidase, to study the role of this enzyme system in vivo. In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure from 105+/-2 to 151+/-6 mm Hg and increased vascular O2*- formation 2- to 3-fold in wild-type (WT) mice. In contrast, in p47(phox-/-) mice the hypertensive response to angiotensin II infusion (122+/-4 mm Hg; P<0.05) was markedly blunted, and there was no increase of vascular O2*- production. In situ staining for O2*- using dihydroethidium revealed a marked increase of O2*-production in both endothelial and vascular smooth muscle cells of angiotensin II-treated WT mice, but not in those of p47(phox-/-) mice. To directly examine the role of the NAD(P)H oxidase in endothelial production of O2*-, endothelial cells from WT and p47(phox-/-) mice were cultured. Western blotting confirmed the absence of p47(phox) in p47(phox-/-) mice. Angiotensin II increased O2*- production in endothelial cells from WT mice, but not in those from p47(phox-/-) mice, as determined by electron spin resonance spectroscopy. These results suggest a pivotal role of the NAD(P)H oxidase and its subunit p47(phox) in the vascular oxidant stress and the blood pressure response to angiotensin II in vivo.

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...

. Shear stress stimulates phosphorylation of eNOS at Ser 635 by a protein kinase A-dependent mechanism.

Recently, we demonstrated that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, either 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, increased endothelial nitric oxide (.NO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR-gamma-stimulated endothelial.NO release remain to be defined. Superoxide anion radical (O2-.) combines with .NO to decrease.NO bioavailability. NADPH oxidase, which produces O2-., and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades O2-., thereby contribute to regulation of endothelial cell.NO metabolism. Therefore, we examined the ability of PPAR-gamma ligands to modulate endothelial O2-. metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 microM 15d-PGJ2 or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent O2-. production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ2 or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, gp91phox (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-PGJ2 or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR-gamma ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial.NO production, PPAR-gamma ligands enhance endothelial.NO bioavailability, in part by altering endothelial O2-. metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR-gamma ligands directly alter vascular endothelial function.

The Arabidopsis gene AtCHIP encodes a protein with three tetratricopeptide repeats and a U-box domain, which is structurally similar to the animal CHIP proteins, a new class of E3 ubiquitin ligases. Like animal CHIP proteins, AtCHIP has E3 ubiquitin ligase activity in vitro. AtCHIP is a single-copy gene, and its transcript is up-regulated by several stress conditions such as low and high temperatures. However, increased AtCHIP expression alone was not correlated with increased stress tolerance; in fact, overexpression of AtCHIP in Arabidopsis rendered plants more sensitive to both low-and high-temperature treatments. Higher electrolyte leakage was observed in leaves of AtCHIP overexpression plants after chilling temperature treatment, suggesting that membrane function is likely impaired in these plants under such a condition. These results indicate that AtCHIP plays an important role in plant cellular metabolism under temperature stress conditions.