Osteopontin Activation and Microcalcification in Venous Grafts Can Be Modulated by Dexamethasone.

BACKGROUND: Osteopontin has been implicated in vascular calcification formation and vein graft intimal hyperplasia, and its expression can be triggered by pro-inflammatory activation of cells. The role of osteopontin and the temporal formation of microcalcification in vein grafts is poorly understood with a lack of understanding of the interaction between haemodynamic changes and the activation of osteopontin. METHODS: We used a porcine model of vein interposition grafts, and human long saphenous veins exposed to ex vivo perfusion, to study the activation of osteopontin using polymerase chain reaction, immunostaining, and 18F-sodium fluoride autoradiography. RESULTS: The porcine model showed that osteopontin is active in grafts within 1 week following surgery and demonstrated the presence of microcalcification. A brief pretreatment of long saphenous veins with dexamethasone can suppress osteopontin activation. Prolonged culture of veins after exposure to acute arterial haemodynamics resulted in the formation of microcalcification but this was suppressed by pretreatment with dexamethasone. 18F-sodium fluoride uptake was significantly increased as early as 1 week in both models, and the pretreatment of long saphenous veins with dexamethasone was able to abolish its uptake. CONCLUSIONS: Osteopontin is activated in vein grafts and is associated with microcalcification formation. A brief pretreatment of veins ex vivo with dexamethasone can suppress its activation and associated microcalcification.

The Role of Preservation Solutions upon Saphenous Vein Endothelial Integrity and Function: Systematic Review and UK Practice Survey.

The long saphenous vein is the most used conduit in cardiac surgery, but its long-term patency is limited by vein graft disease (VGD). Endothelial dysfunction is a key driver of VGD; its aetiology is multi-factorial. However emerging evidence identifies vein conduit harvest technique and preservation fluids as causal in their onset and propagation. This study aims to comprehensively review published data on the relationship between preservation solutions, endothelial cell integrity and function, and VGD in human saphenous veins harvested for CABG. The review was registered with PROSPERO (CRD42022358828). Electronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until August 2022. Papers were evaluated in line with registered inclusion and exclusion criteria. Searches identified 13 prospective, controlled studies for inclusion in the analysis. All studies used saline as a control solution. Intervention solutions included heparinised whole blood and saline, DuraGraft, TiProtec, EuroCollins, University of Wisconsin (UoW), buffered, cardioplegic and Pyruvate solutions. Most studies demonstrated that normal saline appears to have negative effects on venous endothelium and the most effective preservation solutions identified in this review were TiProtec and DuraGraft. The most used preservation solutions in the UK are heparinised saline or autologous whole blood. There is substantial heterogeneity both in practice and reporting of trials evaluating vein graft preservation solutions, and the quality of existing evidence is low. There is an unmet need for high quality trials evaluating the potential for these interventions to improve long-term patency in venous bypass grafts.

The Role of Endothelial Cells in the Onset, Development and Modulation of Vein Graft Disease.

Endothelial cells comprise the intimal layer of the vasculature, playing a crucial role in facilitating and regulating aspects such nutrient transport, vascular homeostasis, and inflammatory response. Given the importance of these cells in maintaining a healthy haemodynamic environment, dysfunction of the endothelium is central to a host of vascular diseases and is a key predictor of cardiovascular risk. Of note, endothelial dysfunction is believed to be a key driver for vein graft disease-a pathology in which vein grafts utilised in coronary artery bypass graft surgery develop intimal hyperplasia and accelerated atherosclerosis, resulting in poor long-term patency rates. Activation and denudation of the endothelium following surgical trauma and implantation of the graft encourage a host of immune, inflammatory, and cellular differentiation responses that risk driving the graft to failure. This review aims to provide an overview of the current working knowledge regarding the role of endothelial cells in the onset, development, and modulation of vein graft disease, as well as addressing current surgical and medical management approaches which aim to beneficially modulate endothelial function and improve patient outcomes.

Next-Generation and Single-Cell Sequencing Approaches to Study Atherosclerosis and Vascular Inflammation Pathophysiology: A Systematic Review.

Background and Aims: Atherosclerosis is a chronic inflammatory disease that remains the leading cause of morbidity and mortality worldwide. Despite decades of research into the development and progression of this disease, current management and treatment approaches remain unsatisfactory and further studies are required to understand the exact pathophysiology. This review aims to provide a comprehensive assessment of currently published data utilizing single-cell and next-generation sequencing techniques to identify key cellular and molecular contributions to atherosclerosis and vascular inflammation. Methods: Electronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until February 2022. A narrative synthesis of all included studies was performed for all included studies. Quality assessment and risk of bias analysis was evaluated using the ARRIVE and SYRCLE checklist tools. Results: Thirty-four studies were eligible for narrative synthesis, with 16 articles utilizing single-cell exclusively, 10 utilizing next-generation sequencing and 8 using a combination of these approaches. Studies investigated numerous targets, ranging from exploratory tissue and plaque analysis, cell phenotype investigation and physiological/hemodynamic contributions to disease progression at both the single-cell and whole genome level. A significant area of focus was placed on smooth muscle cell, macrophage, and stem/progenitor contributions to disease, with little focus placed on contributions of other cell types including lymphocytes and endothelial cells. A significant level of heterogeneity exists in the outcomes from single-cell sequencing of similar samples, leading to inter-sample and inter-study variation. Conclusions: Single-cell and next-generation sequencing methodologies offer novel means of elucidating atherosclerosis with significantly higher resolution than previous methodologies. These approaches also show significant potential for translatability into other vascular disease states, by facilitating cell-specific gene expression profiles between disease states. Implementation of these technologies may offer novel approaches to understanding the disease pathophysiology and improving disease prevention, management, and treatment.Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021229960, identifier: CRD42021229960.

Acute shear stress and vein graft disease.

The long saphenous vein is commonly used in cardiac surgery to bypass occluded coronary arteries. Its use is complicated by late stenosis and occlusion due to the development of intimal hyperplasia. It is accepted that intimal hyperplasia is a multifactorial inflammatory process that starts immediately after surgery. The role of acute changes in haemodynamic conditions when the vein is implanted into arterial circulation, especially shear stress, is not fully appreciated. This review provides an overview of intimal hyperplasia and the effect of acute shear stress changes on the activation of pro-inflammatory mediators.

MicroRNA antagonist therapy during normothermic machine perfusion of donor kidneys.

Normothermic machine perfusion (NMP) is a novel clinical approach to overcome the limitations of traditional hypothermic organ preservation. NMP can be used to assess and recondition organs prior to transplant and is the subject of clinical trials in solid organ transplantation. In addition, NMP provides an opportunity to deliver therapeutic agents directly to the organ, thus avoiding many limitations associated with systemic treatment of the recipient. We report the delivery of oligonucleotide-based therapy to human kidneys during NMP, in this case to target microRNA function (antagomir). An antagomir targeting mir-24-3p localized to the endothelium and proximal tubular epithelium. Endosomal uptake during NMP conditions facilitated antagomir co-localization with proteins involved in the RNA-induced silencing complex (RISC) and demonstrated engagement of the miRNA target. This pattern of uptake was not seen during cold perfusion. Targeting mir-24-3p action increased expression of genes controlled by this microRNA, including heme oxygenase-1 and sphingosine-1-phosphate receptor 1. The expression of genes not under the control of mir-24-3p was unchanged, indicating specificity of the antagomir effect. In summary, this is the first report of ex vivo gymnotic delivery of oligonucleotide to the human kidney and demonstrates that NMP provides the platform to bind and block detrimental microRNAs in donor kidneys prior to transplantation.

The Role of miR-200b-3p in Modulating TGF-ß1-induced Injury in Human Bronchial Epithelial Cells.

BACKGROUND: Dysregulation of microRNAs (miRNAs) has been implicated in airway diseases where transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) may contribute to pathophysiology. Our study investigated the role of miRNA-200b in TGF-ß1-induced EMT in human bronchial epithelial cells. METHODS: NanoString nCounter miRNA assay was used to profile miRNA in control versus TGF-ß1 (1, 4, and 24 h) stimulated BEAS-2B cells. Immortalized primary bronchial epithelial cell line (BEAS-2B cells), human primary bronchial epithelial cells (PBECs), and PBECs derived post-lung transplant were transfected with miR-200b-3p mimics and EMT marker expression was examined at RNA and protein level. miRNA target studies were performed and validated using computational tools and luciferase assay. In situ hybridization was done on normal lung tissue to localize miR-200b-3p in airway epithelium. RESULTS: miR-200b-3p was downregulated post-TGF-ß1 treatment compared with control in BEAS-2B. miR-200b-3p mimic transfection before TGF-ß1 stimulation maintained epithelial marker expression and downregulated mesenchymal cell markers at RNA and protein level in BEAS-2B cells and PBECs. Furthermore, miR-200b-3p mimics reversed established TGF-ß1-induced EMT in BEAS-2B cells. miR-200b-3p targets, ZNF532, and ZEB2 were validated as direct targets using luciferase assay. miR-200b-3p mimics suppress TGF-ß1-induced EMT via inhibition of ZNF532 and ZEB2. In situ hybridization showed that miR-200b-3p is expressed in the normal lung epithelium. Additionally, miR-200b-3p mimics inhibit EMT in the presence of TGF-ß1 in PBECs derived from lung allograft. CONCLUSIONS: We provide proof of concept that miR-200b-3p protects airway epithelial cells from EMT. Manipulating miR-200b-3p expression may represent a novel therapeutic modulator in airway pathophysiology.

The potential role of microRNAs in lung allograft rejection.

Lung transplantation (LT) has proven to be successful in carefully selected individuals with end-stage lung disease. However, long-term graft survival post-LT is often hindered by the development of the bronchiolitis obliterans syndrome (BOS). Because BOS represents is a major problem for all LT centers, early identification and prediction of progressive loss of lung function is a major goal. MicroRNAs (miRNAs) play a major role in regulating many cellular functions, including epithelial-to-mesenchymal transition. miRNAs are emerging not only as biomarkers but also as potential therapy. The recognized importance of injured human bronchial epithelium in lung allograft dysfunction indicates that there is a need for research into the potential role of miRNAs. In this we review we summarize published findings in miRNAs implicated in lung and other types of allograft dysfunction and their role in maintaining the phenotype of epithelial cells after transplant injury. We also address potential clinical interventions that involve manipulating miRNA expression that may promote long-term airway integrity and graft survival.