Immobilized Bisphosphonates as Potential Inhibitors of Bioprosthetic Calcification: Effects on Various Xenogeneic Cardiovascular Tissues

Calcification is the major factor limiting the clinical use of bioprostheses. It may be prevented by the immobilization of bisphosphonic compounds (BPs) on the biomaterial. In this study, we assessed the accumulation and structure of calcium phosphate deposits in collagen-rich bovine pericardium (Pe) and elastin-rich porcine aortic wall (Ao) and bovine jugular vein wall (Ve) cross-linked with glutaraldehyde (GA) or diepoxy compound (DE). These tissues were then modified with pamidronic (PAM) acid or 2-(2′-carboxyethylamino)ethylidene-1,1-bisphosphonic (CEABA) acid. Tissue transformations were studied using Fourier-transform infrared spectroscopy. After subcutaneous implantation of the biomaterials in 220 rats, calcification dynamics were examined using atomic absorption spectrophotometry, light microscopy after von Kossa staining, and scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy The calcium content in all GA-cross-linked tissues and DE-cross-linked Ao increased to 100–160 mg/g on day 60 after implantation. BPs prevented the accumulation of phosphates on the surface of all materials and most effectively inhibited calcification in GA-cross-linked Ao and DE-cross-linked Pe. PAM containing -OH in the R1 group was more effective than CEABA containing -H in R1. The calcification-inhibitory effect of BPs may be realized through their ability to block nucleation and prevent the growth of hydroxyapatite crystals.

Mechano-regulated cell–cell signaling in the context of cardiovascular tissue engineering

Cardiovascular tissue engineering (CVTE) aims to create living tissues, with the ability to grow and remodel, as replacements for diseased blood vessels and heart valves. Despite promising results, the (long-term) functionality of these engineered tissues still needs improvement to reach broad clinical application. The functionality of native tissues is ensured by their specific mechanical properties directly arising from tissue organization. We therefore hypothesize that establishing a native-like tissue organization is vital to overcome the limitations of current CVTE approaches. To achieve this aim, a better understanding of the growth and remodeling (G&R) mechanisms of cardiovascular tissues is necessary. Cells are the main mediators of tissue G&R, and their behavior is strongly influenced by both mechanical stimuli and cell–cell signaling. An increasing number of signaling pathways has also been identified as mechanosensitive. As such, they may have a key underlying role in regulating the G&R of tissues in response to mechanical stimuli. A more detailed understanding of mechano-regulated cell–cell signaling may thus be crucial to advance CVTE, as it could inspire new methods to control tissue G&R and improve the organization and functionality of engineered tissues, thereby accelerating clinical translation. In this review, we discuss the organization and biomechanics of native cardiovascular tissues; recent CVTE studies emphasizing the obtained engineered tissue organization; and the interplay between mechanical stimuli, cell behavior, and cell–cell signaling. In addition, we review past contributions of computational models in understanding and predicting mechano-regulated tissue G&R and cell–cell signaling to highlight their potential role in future CVTE strategies.

Cardiovascular Toxicity Induced by Chronic Vincristine Treatment

Vincristine is an effective anticancer agent for treating leukemias, lymphomas, and other solid tumors. Vincristine’s better-known severe side effects include bone marrow depression, hyponatremia, peripheral neuropathy, and gastrointestinal distress. In recent years, cardiovascular damage also has been described during vincristine treatments. However, the vascular toxicity induced by vincristine is little studied. The aim of the present is to evaluate whether these alterations remain after the suspension of chemotherapy treatment (sequelae) and the possible mechanisms involved in this vascular damage. Adult male Wistar rats were used. The animals were divided into four treatment groups: two groups of saline (0.9% NaCl; saline, sequelae saline) and two groups of vincristine (100 μg/kg; vincristine, sequelae vincristine). Saline or vincristine was administered intraperitoneally in two cycles of 5 days each, leaving a rest period between cycles of 2 days. The final cumulative vincristine dose administered was 1 mg/kg. Sequelae groups correspond to 2 weeks after stopping treatment with the antitumor agent. At the end of the different experimental protocols, cardiac and vascular functions were analyzed. Alterations in the expression of different proteins in the cardiovascular tissues were also investigated. Chronic treatment with vincristine did not produce significant changes in basal cardiac function but provoked significant endothelial dysfunction in the aorta and a significant decrease in the mesenteric contractile function. These cardiovascular functional alterations disappeared 2 weeks after the suspension of chemotherapy treatment. Vincristine treatment caused a significant increase in the expression of tumor necrosis factor-alpha (TNFα), endothelial and inducible nitric oxide synthases (eNOS and iNOS), and connexin 43 in cardiac tissue. In the aorta, the chronic treatment with vincristine caused a slight non-significant increase in TNFα expression, a significant increase in eNOS and iNOS, and a significant decrease in connexin 43. After 2 weeks of vincristine treatment (sequelae group), the expression of TNFα increased and eNOS and iNOS expressions disappeared, but a significant decrease in the expression of connexin 43 was still observed in the aorta. In mesenteric arteries, similar data to those found in the aorta were observed. In conclusion, chronic treatment with vincristine causes functional alterations in the vascular function of both conductance and resistance vessels and changes in the expressions of TNFα, eNOS, iNOS, and connexin 43 in cardiovascular tissues, implicating direct toxicity during its treatment. These functional alterations are transitory and disappear after the suspension of its treatment.

ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the “PXE gene” and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

ARSENIC SPECIES BINDING PROTEINS IN CARDIOVASCULAR TISSUES FROM CARDIAC PATIENTS OF CHILE

The intracellular As-protein binding in cytosol and methanol–water extract of the auricle and saphene tissues of As impacted people was evaluated by bidimensional size exclusion FPLC-UV-ICP-MS. The fractionation of cytosol using Superdex, Phenomenex and MonoQ HR 5/5 columns, shows that As is distributed in a wide range of contiguous fractions of each column, being 8, 25, 50 % the percentages of As in the collected fractions, respectively. In the methanol: water extracts a similar study than performed with the cytosol using preparative gel chromatography on Sephadex G-75 and Shephadex G-100 columns and the MonoQ HR 5/5 anion protein exchange was carried out. A very low As (<1 % of total As) and protein contain were found in the different fractions of both SEC fractionating series. A similar As–protein association to that found in the cytosol after fractionating with MonoQ HR 5/5 was observed for auricle and saphene. Inorganic and methylated As speciation in the 20 – 26 cytosol fractions obtained within the Phenomenex column was performed by HPLC–ICP–MS using the Hamilton PRP-X100 column. Only As (III) and As (V) were present and the results obtained shows that the As (III)/As(V) ratio is constant in most cases. Direct evidence of the existence of As–binding peptides in auricle and saphene vein from arsenic impacted human beings has have been obtained which was previously reported by means of novo peptide synthesis.

Harnessing Single-Cell RNA Sequencing to Better Understand How Diseased Cells Behave the Way They Do in Cardiovascular Disease

The transition of healthy arteries and cardiac valves into dense, cell-rich, calcified, and fibrotic tissues is driven by a complex interplay of both cellular and molecular mechanisms. Specific cell types in these cardiovascular tissues become activated following the exposure to systemic stimuli including circulating lipoproteins or inflammatory mediators. This activation induces multiple cascades of events where changes in cell phenotypes and activation of certain receptors may trigger multiple pathways and specific alterations to the transcriptome. Modifications to the transcriptome and proteome can give rise to pathological cell phenotypes and trigger mechanisms that exacerbate inflammation, proliferation, calcification, and recruitment of resident or distant cells. Accumulating evidence suggests that each cell type involved in vascular and valvular diseases is heterogeneous. Single-cell RNA sequencing is a transforming medical research tool that enables the profiling of the unique fingerprints at single-cell levels. Its applications have allowed the construction of cell atlases including the mammalian heart and tissue vasculature and the discovery of new cell types implicated in cardiovascular disease. Recent advances in single-cell RNA sequencing have facilitated the identification of novel resident cell populations that become activated during disease and has allowed tracing the transition of healthy cells into pathological phenotypes. Furthermore, single-cell RNA sequencing has permitted the characterization of heterogeneous cell subpopulations with unique genetic profiles in healthy and pathological cardiovascular tissues. In this review, we highlight the latest groundbreaking research that has improved our understanding of the pathological mechanisms of atherosclerosis and future directions for calcific aortic valve disease.