The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.

Objective-The regulation of AMP-activated protein kinase (AMPK) is implicated in vascular biology because AMPK can phosphorylate endothelial NO synthase (eNOS). In this study, we investigate the regulation of the AMPK-eNOS pathway in vascular endothelial cells (ECs) by shear stress and the activation of aortic AMPK in a mouse model with a high level of voluntary running (High-Runner). Methods and Results-By using flow channels with cultured ECs, AMPK Thr172 phosphorylation was increased with changes of flow rate or pulsatility. The activity of LKB1, the upstream kinase of AMPK, and the phosphorylation of eNOS at Ser1179 were concomitant with AMPK activation responding to changes in flow rate or pulsatility. The blockage of AMPK by a dominant-negative mutant of AMPK inhibited shear stress-induced eNOS Ser1179 phosphorylation and NO production. Furthermore, aortic AMPK activity and level of eNOS phosphorylation were significantly elevated in the aortas of High-Runner mice. Conclusions-Our results suggest that shear stress activates AMPK in ECs, which contributes to elevated eNOS activity and subsequent NO production. Hence, AMPK, in addition to serving as an energy sensor, also plays an important role in regulating vascular tone. Key Words: endothelium Ⅲ AMPK Ⅲ nitric oxide synthase Ⅲ shear stress Ⅲ exercise E ndothelium-derived NO can enhance vascular functions, including vessel relaxation, survival of vascular endothelial cells (ECs), inhibition of platelet aggregation, and attenuation of leukocyte infiltration. 1,2 Impaired NO bioavailability has been suggested as one of the earliest pathophysiological events preceding endothelial dysfunction and contributing to atherosclerosis. 3,4 Shear stress is an important physiological stimulus that enhances the production of NO by ECs. 2,5 An increase in shear stress such as in exercise augments the EC-mediated bioavailability of NO. 6 Endothelial NO synthase (eNOS), the key enzyme for NO production in ECs, is tightly regulated not only at the transcriptional level but also by several post-translational mechanisms. The enhanced phosphorylation of Ser1179 of bovine eNOS (Ser1177 in humans) leads to increased eNOS activity. Mounting evidence has shown that shear stress enhances the phosphorylation of Ser1177/1179. 7-9 Use of the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and LY 294002 has demonstrated that Akt phosphorylates eNOS Ser1177/1179 in response to shear stress. 7,8 However, dominant-negative mutants of Akt were unable to block the shear stress-stimulated Ser1179 phosphorylation. 9 Further, H89, a protein kinase A (PKA) inhibitor, and an adenovirusexpressing PKA inhibitor (PKI) blocked the eNOS Ser1179 phosphorylation, which indicates the involvement of PKA. 9 -12 Functioning as a metabolic master switch, AMP-activated protein kinase (AMPK) senses and regulates the cellular energy status in various cell types. AMPK is activated by several physiological and pathological stresses such as exercise, hypoxia, and nutrient depletion that result in incre...

The normal coronary artery consists of two mechanically distinct layers: intima-media and adventitia. The objective of this study is to establish a two-layer three-dimensional (3-D) stress-strain relation of porcine coronary arteries. Experimental measurements were made by a series of biaxial tests (inflation and axial extension) of intact coronary arteries and, subsequently, their corresponding intima-media or adventitia layer. The Fung-type exponential strain energy function was used to describe the 3-D strain-stress relation for each layer and the intact wall. A genetic algorithm was used to determine the material constants in the Fung-type constitutive equation by curve fitting the experimental data. Because one layer must be sacrificed before the other layer can be tested, the material property of the missing layer was computed from the material constants of the intact vessel and the tested layer. A total of 20 porcine hearts were used: one group of 10 hearts for the left anterior descending artery and another group of 10 hearts for the right coronary artery. Each group was further divided into two subgroups of five specimens tested for the intact wall and the intima-media layer and for the intact wall and the adventitia layer. Our results show statistically significant differences in the material properties of the two layers. The mathematical model was validated by experimental stress-strain data for individual layers. The validated 3-D constitutive model will serve as a foundation for formulation of layer-specific boundary value problems in coronary physiology and cardiology.

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