Atherosclerosis, calcific aortic valve disease (CAVD), and bioprosthetic heart valve degeneration (alternatively termed structural valve deterioration, SVD) represent three diseases affecting distinct components of the circulatory system and their substitutes, yet sharing multiple risk factors and commonly leading to the extraskeletal calcification. Whereas the histopathology of the mentioned disorders is well-described, their ultrastructural pathology is largely obscure due to the lack of appropriate investigation techniques. Employing an original method for sample preparation and the electron microscopy visualisation of calcified cardiovascular tissues, here we revisited the ultrastructural features of lipid retention, macrophage infiltration, intraplaque/intraleaflet haemorrhage, and calcification which are common or unique for the indicated types of cardiovascular disease. Atherosclerotic plaques were notable for the massive accumulation of lipids in the extracellular matrix (ECM), abundant macrophage content, and pronounced neovascularisation associated with blood leakage and calcium deposition. In contrast, CAVD and SVD generally did not require vasculo- or angiogenesis to occur, instead relying on fatigue-induced ECM degradation and the concurrent migration of immune cells. Unlike native tissues, bioprosthetic heart valves contained numerous specialised macrophages and were not capable of the regeneration that underscores ECM integrity as a pivotal factor for SVD prevention. While atherosclerosis, CAVD, and SVD show similar pathogenesis patterns, these disorders demonstrate considerable ultrastructural differences.

Calcium phosphate bions (CPB) are biomimetic mineralo-organic nanoparticles which represent a physiological mechanism regulating the function, transport and disposal of calcium and phosphorus in the human body. We hypothesised that CPB may be pathogenic entities and even a cause of cardiovascular calcification. Here we revealed that CPB isolated from calcified atherosclerotic plaques and artificially synthesised CPB are morphologically and chemically indistinguishable entities. Their formation is accelerated along with the increase in calcium salts-phosphates/serum concentration ratio. Experiments in vitro and in vivo showed that pathogenic effects of CPB are defined by apoptosis-mediated endothelial toxicity but not by direct tissue calcification or functional changes in anti-calcification proteins. Since the factors underlying the formation of CPB and their pathogenic mechanism closely resemble those responsible for atherosclerosis development, further research in this direction may help us to uncover triggers of this disease.

An association between high serum calcium/phosphate and cardiovascular events or death is well-established. However, a mechanistic explanation of this correlation is lacking. Here, we examined the role of calciprotein particles (CPPs), nanoscale bodies forming in the human blood upon its supersaturation with calcium and phosphate, in cardiovascular disease. The serum of patients with coronary artery disease or cerebrovascular disease displayed an increased propensity to form CPPs in combination with elevated ionised calcium as well as reduced albumin levels, altogether indicative of reduced Ca²⁺-binding capacity. Intravenous administration of CPPs to normolipidemic and normotensive Wistar rats provoked intimal hyperplasia and adventitial/perivascular inflammation in both balloon-injured and intact aortas in the absence of other cardiovascular risk factors. Upon the addition to primary human arterial endothelial cells, CPPs induced lysosome-dependent cell death, promoted the release of pro-inflammatory cytokines, stimulated leukocyte adhesion, and triggered endothelial-to-mesenchymal transition. We concluded that CPPs, which are formed in the blood as a result of altered mineral homeostasis, cause endothelial dysfunction and vascular inflammation, thereby contributing to the development of cardiovascular disease.

Calciprotein particles (CPPs), which increasingly arise in the circulation during the disorders of mineral homeostasis, represent a double-edged sword protecting the human organism from extraskeletal calcification but potentially causing endothelial dysfunction. Existing models, however, failed to demonstrate the detrimental action of CPPs on endothelial cells (ECs) under flow. Here, we applied a flow culture system, where human arterial ECs were co-incubated with CPPs for 4 h, and a normolipidemic and normotensive rat model (10 daily intravenous injections of CPPs) to simulate the scenario occurring in vivo in the absence of confounding cardiovascular risk factors. Pathogenic effects of CPPs were investigated by RT-qPCR and Western blotting profiling of the endothelial lysate. CPPs were internalised within 1 h of circulation, inducing adhesion of peripheral blood mononuclear cells to ECs. Molecular profiling revealed that CPPs stimulated the expression of pro-inflammatory cell adhesion molecules VCAM1 and ICAM1 and upregulated transcription factors of endothelial-to-mesenchymal transition (Snail, Slug and Twist1). Furthermore, exposure to CPPs reduced the production of atheroprotective transcription factors KLF2 and KLF4 and led to YAP1 hypophosphorylation, potentially disturbing the mechanisms responsible for the proper endothelial mechanotransduction. Taken together, our results suggest the ability of CPPs to initiate endothelial dysfunction at physiological flow conditions.

The blend of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) has recently been considered promising for vascular tissue engineering. However, it was shown that PHBV/PCL grafts require biofunctionalization to achieve high primary patency rate. Here we compared immobilization of arginine–glycine–aspartic acid (RGD)-containing peptides and the incorporation of vascular endothelial growth factor (VEGF) as two widely established biofunctionalization approaches. Electrospun PHBV/PCL small-diameter grafts with either RGD peptides or VEGF, as well as unmodified grafts were implanted into rat abdominal aortas for 1, 3, 6, and 12 months following histological and immunofluorescence assessment. We detected CD31⁺/CD34⁺/vWF⁺ cells 1 and 3 months postimplantation at the luminal surface of PHBV/PCL/RGD and PHBV/PCL/VEGF, but not in unmodified grafts, with the further observation of CD31⁺CD34⁻vWF⁺ phenotype. These cells were considered as endothelial and produced a collagen-positive layer resembling a basement membrane. Detection of CD31⁺/CD34⁺ cells at the early stages with subsequent loss of CD34 indicated cell adhesion from the bloodstream. Therefore, either conjugation with RGD peptides or the incorporation of VEGF promoted the formation of a functional endothelial cell layer. Furthermore, both modifications increased primary patency rate three-fold. In conclusion, both of these biofunctionalization approaches can be considered as equally efficient for the modification of tissue-engineered vascular grafts.

The combination of a natural hydrophilic polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a synthetic hydrophobic polymer poly(ε-caprolactone) (PCL) is promising for the preparation of biodegradable and biocompatible small-diameter vascular grafts for bypass surgery. However, physico-mechanical properties and endothelialization rate of PHBV/PCL grafts are poor. We suggested that incorporation of vascular endothelial growth factor (VEGF) into PHBV/PCL grafts may improve their physico-mechanical properties and enhance endothelialization. Here we compared morphology, physico-mechanical properties, and in vivo performance of electrospun small-diameter vascular grafts prepared from PHBV/PCL with and without VEGF. Structure of the graft surface and physico-mechanical properties were examined by scanning electron microscopy and universal testing machine, respectively. Grafts were implanted into rat abdominal aorta for 1, 3, and 6 months with the further histological, immunohistochemical, and immunofluorescence examination. PHBV/PCL grafts with and without VEGF were highly porous and consisted mostly of nanoscale and microscale fibers, respectively. Mean pore diameter and mean pore area were significantly lower in PHBV/PCL/VEGF compared to PHBV/PCL grafts (1.47 µm and 10.05 µm2; 2.63 µm and 47.13 µm2, respectively). Durability, elasticity, and stiffness of PHBV/PCL grafts with VEGF were more similar to internal mammary artery compared to those without, particularly 6 months postimplantation. Both qualitative examination and quantitative image analysis showed that three-fourths of PHBV/PCL grafts with VEGF were patent and had many CD31-, CD34-, and vWF-positive cells at their inner surface. However, all PHBV/PCL grafts without VEGF were occluded and had no or a few CD31-positive cells at the inner surface. Therefore, VEGF enhanced endothelialization and improved graft patency at all the time points in a rat abdominal aorta replacement model. In conclusion, PHBV/PCL grafts with VEGF have better biocompatibility and physico-mechanical properties compared to those without. Incorporation of VEGF improves graft patency and accelerates formation of endothelial cell monolayer.