Background

Vascular endothelial cells actively regulate the infiltration of plasma constituents and circulating cells, including leukocytes, from blood to sub-endothelial tissues. This mechanism is generally considered to be a critical step of initiation and progression of atherosclerosis (Mundi et al., 2018). The regulation of vascular permeability is achieved through the coordinated opening and closure of endothelial cell-cell junctions. In several diseased conditions, endogenous agents such as histamine, thrombin, and vascular endothelial growth factors (VEGF) dramatically, but reversibly, alter the function and organization of cell-cell junctional complexes in diverse ways, resulting in various degrees of increase in permeability (Dejana et al., 2008). We recently demonstrated a unique mechanism, by which CD163-positive alternative macrophages [M(Hb)] engulf hemoglobin-haptoglobin complexes at the sites of intra-plaque hemorrhage (IPH), promoting the release of vascular endothelial growth factor (VEGF), which further causes internalization of vascular endothelial cadherin (VE-cad) from inter-cellular adherens junctions, thus increasing vascular permeability and leading to atherosclerotic plaque progression (Guo et al., 2018). In the aforementioned study, we conducted a novel technique to assess vascular permeability of human coronary arteries by using Evans Blue Dye (EBD) solution. In the same study, FITC-dextran was used as an alternative indicator in one-year-old apoE mouse model to demonstrate the intraplaque hemorrhage in brachiocephalic artery, due to the small size of the mouse arteries and dye sensitivity required for confocal imaging.

Among the vast range of blue dyes that were created during the 20^th century, EBD has been the one with the longest biological history since its first application by Herbert McLean Evans in 1914 (Evans and Schulemann, 1914). EBD is an alkaline synthetic bis-azo (benzidine group) with a molecular weight of 961 Da., and high water solubility, allowing the dye to quickly diffuse throughout the blood stream. Most importantly, when the dye is injected intravenously, it has a high affinity for plasma albumin, giving the dye the ability to remain stable and remain distributed throughout the body for a longer time as a result of a slow excretion rate. All the features of EBD allow it to be an extraordinary agent with multiple potential applications in biomedicine as recently reviewed by Linpeng Yao (Yao et al., 2018). These include but are not limited to estimation of plasma volume, identification of tumors and lymph nodes and as a potential marker of vessel permeability by the use of fluorescence when exposed to green light (Hamer et al., 2002). The principles behind the use of EBD in assays to determine vascular permeability lies in the fact that in normal tissues with normal vascular integrity, albumin is not able to migrate out into the interstitium through the vessel wall endothelial layer. This means that in cases of Albumin-EBD complex, the dye would be limited only to circulation. EBD is relatively non-toxic when used in appropriate concentrations. In vivo experiments using both mice and human subjects have demonstrated that when used in excess, albumin reaches its maximum percent of saturation causing vascular leakage and resulting in a rapid bluish discoloration of tissues (Miles and Miles, 1952). In normal conditions, an adequate permeability barrier is maintained through tight cell-to-cell adherens junctions that are strictly controlled by growth factors, cytokines and other molecules (Radu and Chernoff, 2013). However, in pathological conditions where the integrity of the endothelial layer is affected, plasma proteins including albumin, are able to leak out the vessels as may occur in various disease states. The most common pathophysiological event leading to an increase vascular permeability is acute inflammation which may occur when the vascular wall including the endothelial layer is injured. Vasodilatation, increase in blood flow, disruption of endothelial cell junctions and infiltration of leukocytes are the key players in this process. The EBD-Albumin complex can be seen microscopically as basophilic color in interstitial tissues and indicated increased vascular permeability. In our recent study on endothelial barrier dysfunction after drug-eluting stents implantation, EBD perfusion was performed in rabbit iliac artery stenting model to demonstrate the arteries permeability is associated with poor endothelial VE-cadherin/P120 junctions and higher macrophage infiltration (Harari et al., 2018). Given the advantages of EBD, we decided to use the technique to study the human coronary arteries permeability.