Excessive Adventitial and Perivascular Vascularisation Correlates with Vascular Inflammation and Intimal Hyperplasia.

Albeit multiple studies demonstrated that vasa vasorum (VV) have a crucial importance in vascular pathology, the informative markers and metrics of vascular inflammation defining the development of intimal hyperplasia (IH) have been vaguely studied. Here, we employed two rat models (balloon injury of the abdominal aorta and the same intervention optionally complemented with intravenous injections of calciprotein particles) and a clinical scenario (arterial and venous conduits for coronary artery bypass graft (CABG) surgery) to investigate the pathophysiological interconnections among VV, myeloperoxidase-positive (MPO+) clusters, and IH. We found that the amounts of VV and MPO+ clusters were strongly correlated; further, MPO+ clusters density was significantly associated with balloon-induced IH and increased at calciprotein particle-provoked endothelial dysfunction. Likewise, number and density of VV correlated with IH in bypass grafts for CABG surgery at the pre-intervention stage and were higher in venous conduits which more frequently suffered from IH as compared with arterial grafts. Collectively, our results underline the pathophysiological importance of excessive VV upon the vascular injury or at the exposure to cardiovascular risk factors, highlight MPO+ clusters as an informative marker of adventitial and perivascular inflammation, and propose another mechanistic explanation of a higher long-term patency of arterial grafts upon the CABG surgery.

EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues.

Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope.