Atherosclerosis is closely associated with disturbed flow characterized by low and oscillatory shear stress, but studies directly linking disturbed flow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which disturbed flow can be acutely induced and cause atherosclerosis. Here, we characterize partial carotid ligation as a model of disturbed flow with characteristics of low and oscillatory wall shear stress. We also describe a method of isolating intimal RNA in sufficient quantity from mouse carotid arteries. Using this model and method, we found that partial ligation causes upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and rapid atherosclerosis in 2 wk in a p47^phox-dependent manner and advanced lesions by 4 wk. We found that partial ligation results in endothelial dysfunction, rapid atherosclerosis, and advanced lesion development in a physiologically relevant model of disturbed flow. It also allows for easy and rapid intimal RNA isolation. This novel model and method could be used for genome-wide studies to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases.

Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of atherosclerotic lesions, which develop at regions of arteries that are exposed to complex blood flow. In addition, plaque progression and eventually plaque rupture is influenced by a complex interaction between biological and mechanical factors—mechanical forces regulate the cellular and molecular composition of plaques and, conversely, the composition of plaques determines their ability to withstand mechanical load. A deeper understanding of these interactions is essential for designing new therapeutic strategies to prevent lesion development and promote plaque stabilization. Moreover, integrating clinical imaging techniques with finite element modelling techniques allows for detailed examination of local morphological and biomechanical characteristics of atherosclerotic lesions that may be of help in prediction of future events. In this ESC Position Paper on biomechanical factors in atherosclerosis, we summarize the current ‘state of the art’ on the interface between mechanical forces and atherosclerotic plaque biology and identify potential clinical applications and key questions for future research.

Hypertension caused by angiotensin II is dependent on vascular superoxide (O ₂ ·−) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O ₂ ·− 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 O ₂ ·− 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 O ₂ ·− production. In situ staining for O ₂ ·− using dihydroethidium revealed a marked increase of O ₂ ·−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 O ₂ ·−, 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 O ₂ ·− 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.

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 ([ ]) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in [ ] was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H₂O₂production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent [ ] 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 p47^phoxsubunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed [ ] production and had minimal XO protein and activity. Transfection of these cells with p47^phoxrestored 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 [ ] 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.

Recently, we have shown that shear stress stimulates NO^⋅ production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574–1581). Akt has been believed to regulate shear-dependent production of NO^⋅ by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser¹¹⁷⁹ residue (eNOS-S¹¹⁷⁹), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt^AA or Akt^AAA) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S¹¹⁷⁹. Here, we show that shear-dependent phosphorylation of eNOS-S¹¹⁷⁹ is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt^AAor Akt^AAA in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S¹¹⁷⁹ if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt^AAinhibited shear-dependent NO^⋅ production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S¹¹⁷⁹and NO^⋅ production. In contrast, H89 did not inhibit phosphorylation of eNOS-S¹¹⁷⁹ induced by expressing a constitutively active Akt mutant (Akt^Myr) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S¹¹⁷⁹ within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S¹¹⁷⁹ in a PKA-dependent, but Aktindependent manner, whereas the NO^⋅ production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.