Hemodynamic manipulation of eNOS in vivo

Comment on Cheng et al, page 3691

In this issue of Blood, Cheng and colleagues at Erasmus University in Rotterdam demonstrate that arterial eNOS expression in eNOS-GFP transgenic mice is responsive both to hemodynamic flow characteristics and to the magnitude of shear stress forces (τ). These experiments provide important in vivo validation of numerous in vitro studies of the flow-eNOS relationships and reveal the spatial sensitivity of hemodynamic characteristics in the regulation of eNOS in intact arteries.

Hemodynamic regulation of vascular physiology occurs through convective mass transport and through forces imparted to the vessel wall. Flow is also a pathologic determinant of the localization of atherosclerotic lesions that originate at sites of geometric (and hemodynamic) complexity. A particular target of flow forces is the arterial endothelium, an interface that is sensitive to the local (frictional) shear stress.¹  An important physiologic example of τ-endothelial interactions is flow-mediated generation of nitric oxide (NO) by activation of endothelial nitric oxide synthase (eNOS). Regulation of this enzyme is considered to be of major physiologic and pathologic importance. Its activities play a dominant role in vasodilatation by relaxing the smooth muscle cells of arteries to reduce blood pressure. NO also inhibits platelet aggregation, leukocyte adhesion to the endothelium, and cell migration. The effects of τ on eNOS transcription, translation, posttranslational activation, and subcellular compartmentalization have been extensively studied in cultured endothelial cells using simple flow protocols.²  Other in vitro flow studies have attempted to simulate the complex flow characteristics associated with sites of lesion susceptibility in the arterial circulation.³  Such regions correlate with decreased eNOS transcript expression even in lesion-free animals⁴  but the relationship between cause (complex flow) and effect (eNOS expression and localization) had not been established in vivo.

To address these issues, the Rotterdam group generated transgenic mice that express human eNOS in fusion with green fluorescent protein (GFP) as a reporter of eNOS protein expression.⁵  To demonstrate hemodynamic cause-effect, gradations of τ and separations of flow were created by placement of a tapered cast around the midportion of the left common carotid artery, resulting in a corresponding gradual narrowing of the artery lumen over a length of several millimeters. This is normally a region of pulsatile laminar flow without flow separation and where τ forces are unidirectional. It is also a site resistant to atherosclerotic lesion development. Since τ is proportional to 1/(diameter)³ , shear stress increases rapidly throughout the length of the cast. Downstream of the cast, the lumen widens to create a short region of oscillating separated flow similar to that recorded at atherosclerosis-susceptible locations elsewhere. Within 24 hours following placement, the hemodynamics were spatially mapped to face cell responses. Cheng and colleagues report that eNOS gene and protein expression was elevated as τ increased within the tapered cast consistent with shear stress experiments in vitro and that the intracellular redistribution of eNOS, including its activated form (serine 1177 phosphorylation), was significantly increased both by elevated τ and oscillatory flow, although the fraction of total eNOS that was phosphorylated remained unaltered.

The studies are encouraging for investigations both in vivo and in vitro. They suggest that flow-related mechanisms of eNOS regulation identified in reductionist experiments in tissue culture also occur in vivo, albeit with subtleties imparted by a more complex environment. Furthermore, studies of flow disturbance in relation to localized endothelial phenotype in vivo can now be cautiously interpreted as cause-and-effect mechanisms rather than simply correlative. ▪