Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

Abstract

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

Guidelines for the measurement of vascular function and structure in isolated arteries and veins

“…This should allow for maintenance of the intracellular (cytoskeletal) and extracellular matrix structures that affect the mechanical properties of the vascular wall. Indeed, it has been shown that cytoskeletal structures, such as Factin stress fibers, as well as extracellular matrix components, such as elastin and collagen, participate in providing mechanical strength to the vascular wall under passive conditions, albeit each at different intravascular pressures (113)(114)(115)(116)(117)(118)(119)(120). In addition, to avoid a major involvement of the axial component in the strain and stress assessment, vessels should be extended to eliminate any bending or bowing at the maximal intraluminal pressure used.…”

“…Having two measures of vascular stiffness at complementary regions of the pressure-diameter curve allows for determination of the partial contribution that elements such as elastin and collagen play in the mechanical properties of the vascular wall (122). At low pressures, elastin is considered to be the dominant element, whereas at higher pressures collagen is dominant (114,115). Therefore, calculation of the low modulus of elasticity (E low , also known as E elastin ) at low pressures, and the high modulus of elasticity (E high , also known as E collagen ) at higher pressures, estimates the partial contribution of elastin and collagen to the mechanical properties of the vessel wall.…”

An analysis modality for vascular structures combining tissue-clearing technology and topological data analysis

“…Recently, some studies describing signal segmentation of vasculatures utilizing machine learning have been reported 63,64 . Ilastik software is a helpful segmentation tool and is used for counting cell numbers or monitoring the amounts extracellular matrix (ECM) such as elastin 42,43,65 . Currently, measuring volumes or counting the dividing points remains the primary method for quantification of vasculatures.…”

“…Tiff images with original signal intensities were captured by light sheet fluorescent microscopy (LSFM), and these tiff images were converted into hdf5 files with python. Signals were classified using ilastik software as previously described [42][43][44] (Fig. 3a).…”

Endogenous vascular repair system in cardiovascular disease: The role of endothelial progenitor cells

“…5 Finally, rigid vascular wall with altered vasodilation including capillary rarefaction and increased aortic diameter are usually described as core components of vascular remodelling accompanying with nature evolution of the CV disease. 6 Vascular remodelling is triggered by wide spectrum of stimuli, such as mechanical (high blood pressure, shear stress), metabolic (lipids, hyperglycaemia, hyperuricemia, hyper-and hypothyroidisms, hyperphosphatemia), inflammatory (reactive oxygen species, inflammatory cytokines and chemokines), ischemia / hypoxia that may also close associate with senescence and vascular aging. [7][8][9] There is large body of evidence of triggers able to epigenetically modify precursors, a process by which cells stop proliferating and become dysfunctional.…”