Indentation measurements of the subendothelial matrix in bovine carotid arteries

Artery biomechanics are an important factor in cardiovascular function and atherosclerosis development; as such, the macro-mechanics of whole arteries are well-characterized. However, much less is known about the mechanical properties of individual layers in the blood vessel wall. Since there is significant evidence to show that cells can sense the mechanical properties of their matrix, it is critical to characterize the mechanical properties of these individual layers at the scale sensed by cells. Here, we measured subendothelium mechanics in bovine carotid arteries using atomic force microscopy (AFM) indentation. To specifically indent the subendothelium, we evaluated three potential de-endothelialization methods: scraping, paper imprinting, and saponin incubation. Using scanning electron microscopy, histology stains, immunohistochemistry, and multiphoton microscopy, we found that scraping was the only effective de-endothelialization method capable of removing endothelial cells and leaving the subendothelial matrix largely intact. To determine the indentation modulus of the subendothelial matrix, both untreated and scraped (de-endothelialized) bovine carotid arteries were indented with a spherical AFM probe and the data were fit using the Hertz model. Both the endothelium on the untreated artery and the en face subendothelium had similar indentation moduli: E=2.5±1.9 and 2.7±1.1 kPa, respectively. These measurements are the first to quantify the micro-scale mechanics of the subendothelial layer, and constitute a critical step in understanding the relationship between altered subendothelial micromechanics and disease progression.