Preserving Endothelial Integrity in Human Saphenous Veins during Preparation for Coronary Bypass Surgery.

INTRODUCTION: While multiple factors influence coronary artery bypass graft (CABG) success rates, preserving saphenous vein endothelium during surgery may improve patency. Standard preparations include saphenous vein preparation in heparinized saline (saline) which can result in endothelial loss and damage. Here, we investigated the impact of preparing saphenous graft vessels in heparinized patient blood (blood) versus saline. METHODS: Saphenous vein tissues from a total of 23 patients undergoing CABG were split into 2 groups (1) saline and (2) heparinized patient blood. Excess tissue was fixed for analysis immediately following surgery. Level of endothelial coverage, oxidative stress marker 4-hydroxynonenal (4HNE), and oxidative stress protective marker nuclear factor erythroid 2-related factor 2 (NRF2) were evaluated. RESULTS: In saline patient veins, histological analysis revealed a limited luminal layer, suggesting a loss of endothelial cells (ECs). Immunofluorescent staining of EC markers vascular endothelial cadherin (VE-cadherin) and endothelial nitric oxide identified a significant improvement in EC coverage in the blood versus saline groups. Although both treatment groups expressed 4HNE to similar levels, EC blood samples expressed higher levels of NRF2. CONCLUSION: Our data indicate that use of heparinized patient blood helps preserve the endothelium and promotes vein graft health. This has the potential to improve long-term outcomes in patients.

Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity.

AIMS: Calcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored. METHODS AND RESULTS: An aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype. CONCLUSION: Sortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Connexin 43 across the Vasculature: Gap Junctions and Beyond.

Connexin 43 (Cx43) is essential to the function of the vasculature. Cx43 proteins form gap junctions that allow for the exchange of ions and molecules between vascular cells to facilitate cell-to-cell signaling and coordinate vasomotor activity. Cx43 also has intracellular signaling functions that influence vascular cell proliferation and migration. Cx43 is expressed in all vascular cell types, although its expression and function vary by vessel size and location. This includes expression in vascular smooth muscle cells (vSMC), endothelial cells (EC), and pericytes. Cx43 is thought to coordinate homocellular signaling within EC and vSMC. Cx43 gap junctions also function as conduits between different cell types (heterocellular signaling), between EC and vSMC at the myoendothelial junction, and between pericyte and EC in capillaries. Alterations in Cx43 expression, localization, and post-translational modification have been identified in vascular disease states, including atherosclerosis, hypertension, and diabetes. In this review, we discuss the current understanding of Cx43 localization and function in healthy and diseased blood vessels across all vascular beds.

ApoC-III is a novel inducer of calcification in human aortic valves.

Calcific aortic valve disease (CAVD) occurs when subpopulations of valve cells undergo specific differentiation pathways, promoting tissue fibrosis and calcification. Lipoprotein particles carry oxidized lipids that promote valvular disease, but low-density lipoprotein-lowering therapies have failed in clinical trials, and there are currently no pharmacological interventions available for this disease. Apolipoproteins are known promoters of atherosclerosis, but whether they possess pathogenic properties in CAVD is less clear. To search for a possible link, we assessed 12 apolipoproteins in nonfibrotic/noncalcific and fibrotic/calcific aortic valve tissues by proteomics and immunohistochemistry to understand if they were enriched in calcified areas. Eight apolipoproteins (apoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoL-I, and apoM) were enriched in the calcific versus nonfibrotic/noncalcific tissues. Apo(a), apoB, apoC-III, apoE, and apoJ localized within the disease-prone fibrosa and colocalized with calcific regions as detected by immunohistochemistry. Circulating apoC-III on lipoprotein(a) is a potential biomarker of aortic stenosis incidence and progression, but whether apoC-III also induces aortic valve calcification is unknown. We found that apoC-III was increased in fibrotic and calcific tissues and observed within the calcification-prone fibrosa layer as well as around calcification. In addition, we showed that apoC-III induced calcification in primary human valvular cell cultures via a mitochondrial dysfunction/inflammation-mediated pathway. This study provides a first assessment of a broad array of apolipoproteins in CAVD tissues, demonstrates that specific apolipoproteins associate with valvular calcification, and implicates apoC-III as an active and modifiable driver of CAVD beyond its potential role as a biomarker.

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.

Glucose withdrawal induces Endothelin 1 release with significant angiogenic effect from first trimester (FTM), but not term human umbilical cord perivascular cells (HUCPVC).

BACKGROUND: Perivascular cells (PVC) and their "progeny," mesenchymal stromal cells (MSC), have high therapeutic potential for ischemic diseases. While hypoxia can increase their angiogenic properties, the other aspect of ischemic conditions-glucose shortage-is deleterious for MSC and limits their therapeutic applicability. Regenerative cells in developing vascular tissues, however, can adapt to varying glucose environment and react in a tissue-protective manner. Placental development and fetal insulin production generate different glucose fluxes in early and late extraembryonic tissues. We hypothesized that FTM HUCPVC, which are isolated from a developing vascular tissue with varying glucose availability react to low-glucose conditions in a pro-angiogenic manner in vitro. METHODS: Xeno-free (Human Platelet Lysate 2.5%) expanded FTM (n = 3) and term (n = 3) HUCPVC lines were cultured in low (2 mM) and regular (4 mM) glucose conditions. After 72 h, the expression (Next Generation Sequencing) and secretion (Proteome Profiler) of angiogenic factors and the functional angiogenic effect (rat aortic ring assay and Matrigel™ plug) of the conditioned media were quantified and statistically compared between all cultures. RESULTS: Low-glucose conditions had a significant post-transcriptional inductive effect on FTM HUCPVC angiogenic factor secretion, resulting in significantly higher VEGFc and Endothelin 1 release in 3 days compared to term counterparts. Conditioned media from low-glucose FTM HUCPVC cultures had a significantly higher endothelial network enhancing effect compared to all other experimental groups both in vitro aortic ring assay and in subcutan Matrigel™ plugs. Endothelin 1 depletion of the low-glucose FTM HUCPVC conditioned media significantly diminished its angiogenic effect CONCLUSIONS: FTM HUCPVC isolated from an early extraembryonic tissue show significant pro-angiogenic paracrine reaction in low-glucose conditions at least in part through the excess release of Endothelin 1. This can be a substantial advantage in cell therapy applications for ischemic injuries.

Combination human umbilical cord perivascular and endothelial colony forming cell therapy for ischemic cardiac injury.

Cell-based therapeutics are promising interventions to repair ischemic cardiac tissue. However, no single cell type has yet been found to be both specialized and versatile enough to heal the heart. The synergistic effects of two regenerative cell types including endothelial colony forming cells (ECFC) and first-trimester human umbilical cord perivascular cells (FTM HUCPVC) with endothelial cell and pericyte properties respectively, on angiogenic and regenerative properties were tested in a rat model of myocardial infarction (MI), in vitro tube formation and Matrigel plug assay. The combination of FTM HUCPVCs and ECFCs synergistically reduced fibrosis and cardiomyocyte apoptosis, while promoting favorable cardiac remodeling and contractility. These effects were in part mediated by ANGPT2, PDGF-ß, and VEGF-C. PDGF-ß signaling-dependent synergistic effects on angiogenesis were also observed in vitro and in vivo. FTM HUCPVCs and ECFCs represent a cell combination therapy for promoting and sustaining vascularization following ischemic cardiac injury.

A solution to prevent secondary flow in adherent cell cultures.

High quality cell cultures require reliable laboratory practices. Today's small-scale in vitro cell culture format is dominated by circular topology vessels, with the inherent disadvantage of secondary flow induced each time the cell cultures are repositioned. The secondary flow generates uneven sedimentation and adherence that negatively impacts cell culture quality. Here we show a modification of the circular culture vessel that abrogates these disturbances. Cell culture wells were augmented with a central column to diminish secondary flow. Human carcinoma cell lines (BeWo, JEG-3), mesenchymal stem cells [human umbilical cord perivascular cells (HUCPVC)] and mouse embryonic fibroblasts (MEF) were cultured in both column-augmented and regular culture wells. Human carcinoma cell cultures showed even cell densities and significantly more viable cells in column-augmented vessels. In FTM HUCPVC cultures, cell surface MSC marker (CD90, CD105) expression and cell differentiation-related gene expression patterns were significantly more homogeneous in column-augmented vessels. MEF cells in column-augmented culture vessels showed a more consistent expression of IGF-1. Column-augmented cell culture vessels significantly improve the homogeneity of adherent cell cultures by mitigating the adverse effect of the secondary flow.This article has an associated First Person interview with the first author of the paper.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

Angiogenic potency evaluation of cell therapy candidates by a novel application of the in vitro aortic ring assay.

BACKGROUND: Due to limitations of current angiogenesis assays, we aimed to develop a novel application of the rat aortic ring assay to assess the angiogenic potential of mesenchymal stromal cells (MSCs). First-trimester human umbilical cord-derived perivascular cells (FTM HUCPVCs) have multipotent characteristics and previously demonstrated angiogenic potential. We compared the effect of this young source of MSCs and adult bone marrow stromal cells (BMSCs) on ex vivo aortic endothelial network formation. METHODS: Thoracic segments of adult rat aortas were isolated, sectioned and embedded into Matrigel™. Fluorophore-labeled FTM HUCPVC lines and BMSCs (N = 3) were cocultured with developing endothelial networks (day 0). MSC integration, tube formation and endothelial network growth were monitored daily using phase-contrast and fluorescence microscopy. Quantification of endothelial networks was performed using ImageJ network analysis software on day 5 of coculture. RESULTS: FTM HUCPVCs from two umbilical cord samples migrated toward and integrated with developing aortic ring tubular networks while displaying elongated morphologies (day 1). In contrast, BMSCs did not show targeted migration and maintained spherical morphologies with limited physical interactions. Within 1 week of coculture, FTM HUCPVC lines contributed to significantly greater radial network growth and network loop formation when compared to BMSCs and untreated networks. CONCLUSIONS: We have developed a novel potency assay to assess the angiogenic potential of cell therapy candidates. Favorable properties of FTM HUCPVCs over BMSCs that we observed with this assay and which merit further study include chemotaxis, affinity for developing vasculature, and physical supportive interactions contributing to the development of endothelial networks.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature. Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and "translatable" pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection. We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

In Vitro Differentiation of First Trimester Human Umbilical Cord Perivascular Cells into Contracting Cardiomyocyte-Like Cells.

Myocardial infarction (MI) causes an extensive loss of heart muscle cells and leads to congestive heart disease (CAD), the leading cause of mortality and morbidity worldwide. Mesenchymal stromal cell- (MSC-) based cell therapy is a promising option to replace invasive interventions. However the optimal cell type providing significant cardiac regeneration after MI is yet to be found. The aim of our study was to investigate the cardiomyogenic differentiation potential of first trimester human umbilical cord perivascular cells (FTM HUCPVCs), a novel, young source of immunoprivileged mesenchymal stromal cells. Based on the expression of cardiomyocyte markers (cTnT, MYH6, SIRPA, and CX43) FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to bone marrow MSCs, while their immunogenicity remained significantly lower as indicated by HLA-A and HLA-G expression and susceptibility to T cell mediated cytotoxicity. When applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells within 1 week of coculture, making them the first MSC type with this ability. Our results indicate that young FTM HUCPVCs have superior cardiomyogenic potential coupled with beneficial immunogenic properties when compared to MSCs of older tissue sources, suggesting that in vitro predifferentiation could be a potential strategy to increase their effectiveness in vivo.