Microstructure and mechanics of human resistance arteries

Abstract

Vascular diseases such as diabetes and hypertension cause changes to the vasculature that can lead to vessel stiffening and the loss of vasoactivity. The microstructural bases of these changes are not presently fully understood. We present a new methodology for stain-free visualization, at a microscopic scale, of the morphology of the main passive components of the walls of unfixed resistance arteries and their response to changes in transmural pressure. Human resistance arteries were dissected from subcutaneous fat biopsies, mounted on a perfusion myograph, and imaged at varying transmural pressures using a multimodal nonlinear microscope. High-resolution three-dimensional images of elastic fibers, collagen, and cell nuclei were constructed. The honeycomb structure of the elastic fibers comprising the internal elastic layer became visible at a transmural pressure of 30 mmHg. The adventitia, comprising wavy collagen fibers punctuated by straight elastic fibers, thinned under pressure as the collagen network straightened and pulled taut. Quantitative measurements of fiber orientation were made as a function of pressure. A multilayer analytical model was used to calculate the stiffness and stress in each layer. The adventitia was calculated to be up to 10 times as stiff as the media and experienced up to 8 times the stress, depending on lumen diameter. This work reveals that pressure-induced reorganization of fibrous proteins gives rise to very high local strain fields and highlights the unique mechanical roles of both fibrous networks. It thereby provides a basis for understanding the micromechanical significance of structural changes that occur with age and disease.

Keywords: blood pressure; extracellular matrix; mechanical modeling; resistance artery; stress.

Fig. 1.

False color images of a 138-μm-lumen-diameter vessel at 3 mmHg transmural pressure showing second-harmonic generation (SHG, collagen) in blue and two-photon fluorescence (TPF, elastic fibers and cellular fluorescence) in green. Red labels: adventitia (A), media (M), and intima (I). A: optical section through the adventitia showing wavy collagen punctuated by thin elastic fibers. B: reconstruction of a section along the central vessel axis showing thick longitudinally aligned elastic fibers of the internal elastic layer (IEL). C: axial section. D: 3-dimensional (3D) section of the imaged region of the vessel. Bars, 50 μm.

Fig. 2.

False color images of the vessel pictured in Fig. 1 raised to a transmural pressure of 30 mmHg causing the lumen to dilate to a diameter of 172 μm. A: optical section through the adventitia showing a visually relatively unchanged fibrous matrix. B: reconstruction of a section along the central vessel axis. Gaps caused by intimal dilation appear between the elastic fibers of the IEL, which in places bulge apart, braced by thinner connecting fibers. C: axial section. D: 3D section of the imaged region of the vessel. Bars, 50 μm.

Fig. 3.

False color images of the distribution of elastin (green), collagen (blue), and cell nuclei stained with 4′,6-diamidino-2-phenylindole dilactate (DAPI, red) of a vessel at 30 mmHg transmural pressure with a 268-μm lumen diameter. Yellow labels: adventitia, media, and intima. A: section through the adventitia showing adventitial textured collagen and cell nuclei. B: section through the adventitia and media showing slender vascular smooth muscle cell (VSMC) nuclei. C: section through the adventitia, media, and intima showing the IEL and longitudinally aligned endothelial nuclei. D: section through the wall and lumen. Bars, 50 μm.

Fig. 4.

Variations in morphology of vessels. A: axial optical section of a large vessel exhibiting wall thinning (arrow). B: optical section of adventitia of an atypical vessel with significantly increased elastic fiber content. C: axial optical section through a vessel undergoing myogenic contraction, exhibiting a crinkled IEL. D: high-zoom TPF image of an elastin fiber located between two VSMCs. Bars = 50 (A–C) and 25 (D) μm.

Fig. 5.

Mechanics data. A: elastic moduli assuming a layered vessel wall [media modulus (E_m), adventitia stiffness (E_a)] and a homogeneous wall (E_h). Statistical significance: *P < 0.05 and **P < 0.01. B: volumetric strains in the vessel walls. C: peak circumferential stress in each layer analyzed for the vessel depicted in Figs. 1 and 2, with increasing transmural pressure. D: ratio of adventital circumferential stress (σ_θa) to medial circumferential stress (σ_θm) at 30 mmHg transmural pressure with increasing lumen diameter. The positive trend indicates that the adventitia experiences proportionally greater wall stress in larger vessels.

Fig. 6.

Orientation distributions of the three fibrous networks.