Targeting Lipoprotein(a): Can RNA Therapeutics Provide the Next Step in the Prevention of Cardiovascular Disease?

Numerous genetic and epidemiologic studies have demonstrated an association between elevated levels of lipoprotein(a) (Lp[a]) and cardiovascular disease. As a result, lowering Lp(a) levels is widely recognized as a promising strategy for reducing the risk of new-onset coronary heart disease, stroke, and heart failure. Lp(a) consists of a low-density lipoprotein-like particle with covalently linked apolipoprotein A (apo[a]) and apolipoprotein B-100, which explains its pro-thrombotic, pro-inflammatory, and pro-atherogenic properties. Lp(a) serum concentrations are genetically determined by the apo(a) isoform, with shorter isoforms having a higher rate of particle synthesis. To date, there are no approved pharmacological therapies that effectively reduce Lp(a) levels. Promising treatment approaches targeting apo(a) expression include RNA-based drugs such as pelacarsen, olpasiran, SLN360, and lepodisiran, which are currently in clinical trials. In this comprehensive review, we provide a detailed overview of RNA-based therapeutic approaches and discuss the recent advances and challenges of RNA therapeutics specifically designed to reduce Lp(a) levels and thus the risk of cardiovascular disease.

Endothelial damage inhibitor preserves the integrity of venous endothelial cells from patients undergoing coronary bypass surgery.

OBJECTIVES: Despite the success of coronary artery bypass graft (CABG) surgery using autologous saphenous vein grafts (SVGs), nearly 50% of patients experience vein graft disease within 10 years of surgery. One contributing factor to early vein graft disease is endothelial damage during short-term storage of SVGs in inappropriate solutions. Our aim was to evaluate the effects of a novel endothelial damage inhibitor (EDI) on SVGs from patients undergoing elective CABG surgery and on venous endothelial cells (VECs) derived from these SVGs. METHODS: SVGs from 11 patients participating in an ongoing clinical registry (NCT02922088) were included in this study, and incubated with both full electrolyte solution (FES) or EDI for 1 h and then examined histologically. In 8 of 11 patients, VECs were isolated from untreated grafts, incubated with both FES and EDI for 2 h under hypothermic stress conditions and then analysed for activation of an inflammatory phenotype, cell damage and cytotoxicity, as well as endothelial integrity and barrier function. RESULTS: The EDI was superior to FES in protecting the endothelium in SVGs (74 ± 8% versus 56 ± 8%, P < 0.001). Besides confirming that the EDI prevents apoptosis in SVG-derived VECs, we also showed that the EDI temporarily reduces adherens junctions in VECs while protecting focal adhesions compared to FES. CONCLUSIONS: The EDI protects the connectivity and function of the SVG endothelium. Our data suggest that the EDI can preserve focal adhesions in VECs during short-term storage after graft harvesting. This might explain the superiority of the EDI in maintaining most of the endothelium in venous CABG surgery conduits.

Treatment of Cardiac Fibrosis with Extracellular Vesicles: What Is Missing for Clinical Translation?

Heart failure is the leading cause of morbidity and mortality and currently affects more than 60 million people worldwide. A key feature in the pathogenesis of almost all forms of heart failure is cardiac fibrosis, which is characterized by excessive accumulation of extracellular matrix components in the heart. Although cardiac fibrosis is beneficial in the short term after acute myocardial injury to preserve the structural and functional integrity of the heart, persistent cardiac fibrosis contributes to pathological cardiac remodeling, leading to mechanical and electrical dysfunction of the heart. Despite its high prevalence, standard therapies specifically targeting cardiac fibrosis are not yet available. Cell-based approaches have been extensively studied as potential treatments for cardiac fibrosis, but several challenges have been identified during clinical translation. The observation that extracellular vesicles (EVs) derived from stem and progenitor cells exhibit some of the therapeutic effects of the parent cells has paved the way to overcome limitations associated with cell therapy. However, to make EV-based products a reality, standardized methods for EV production, isolation, characterization, and storage must be established, along with concrete evidence of their safety and efficacy in clinical trials. This article discusses EVs as novel therapeutics for cardiac fibrosis from a translational perspective.

Relationship between epicardial adipose tissue attenuation and coronary artery disease in type 2 diabetes mellitus patients.

BACKGROUND AND AIMS: High epicardial adipose tissue (EAT) attenuation is a key characteristic of adipose tissue dysfunction and associated with coronary artery disease (CAD). As little is known about the modulation of EAT attenuation by metabolic disorders, we investigated the association between EAT attenuation and CAD risk factors, CAD presence and CAD severity in type 2 diabetes mellitus (T2DM) patients. METHODS: We included 276 inpatients with T2DM and 305 control patients with normal glucose metabolism (NGM), who underwent cardiac computed tomography angiography (CCTA) and coronary artery calcium (CAC) scoring. EAT attenuation and volume were evaluated by contrast-enhanced CCTA image analysis. Furthermore, segment stenosis scores (SSSs) of the left main coronary artery (LMCA), left anterior descending artery (LAD), left circumflex artery (LCX), right coronary artery (RCA), diagonal/intermediate branch (D/I) and obtuse marginal branch (OM) were calculated to assess CAD severity. RESULTS: T2DM patients showed higher significant CAC scores, coronary plaque prevalence, total SSSs and LMCA-SSSs, LAD-SSSs, LCX-SSSs, RCA-SSSs and D/I-SSSs compared with NGM controls. In contrast to NGM controls, EAT volume was significantly increased in T2DM patients, whereas EAT attenuation was similar. In T2DM patients, EAT attenuation was associated with discrete CAD risk factors, the presence of coronary and triple-vessel plaques, as well as LAD-SSSs, LCX-SSSs, RCA-SSSs and total SSSs. In addition, EAT attenuation was only associated with the total SSS of calcified plaques, but not with noncalcified plaques. CONCLUSION: In T2DM patients, high EAT attenuation is associated with the presence and severity of CAD in general and with coronary stenosis caused by calcified plaques in particular.

Anisotropic topographies restore endothelial monolayer integrity and promote the proliferation of senescent endothelial cells.

Thrombogenicity remains a major issue in cardiovascular implants (CVIs). Complete surficial coverage of CVIs by a monolayer of endothelial cells (ECs) prior to implantation represents a promising strategy but is hampered by the overall logistical complexity and the high number of cells required. Consequently, extensive cell expansion is necessary, which may eventually lead to replicative senescence. Considering that micro-structured surfaces with anisotropic topography may promote endothelialization, we investigated the impact of gratings on the biomechanical properties and the replicative capacity of senescent ECs. After cultivation on gridded surfaces, the cells showed significant improvements in terms of adherens junction integrity, cell elongation, and orientation of the actin filaments, as well as enhanced yes-associated protein nuclear translocation and cell proliferation. Our data therefore suggest that micro-structured surfaces with anisotropic topographies may improve long-term endothelialization of CVIs.

The path to a hemocompatible cardiovascular implant: Advances and challenges of current endothelialization strategies.

Cardiovascular (CV) implants are still associated with thrombogenicity due to insufficient hemocompatibility. Endothelialization of their luminal surface is a promising strategy to increase their hemocompatibility. In this review, we provide a collection of research studies and review articles aiming to summarize the recent efforts on surface modifications of CV implants, including stents, grafts, valves, and ventricular assist devises. We focus in particular on the implementation of micrometer or nanoscale surface modifications, physical characteristics of known biomaterials (such as wetness and stiffness), and surface morphological features (such as gratings, fibers, pores, and pits). We also review how biomechanical signals originating from the endothelial cell for surface interaction can be directed by topography engineering approaches toward the survival of the endothelium and its long-term adaptation. Finally, we summarize the regulatory and economic challenges that may prevent clinical implementation of endothelialized CV implants.

Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems.

MicroRNAs play an essential role in cell homeostasis and have been proposed as therapeutic agents. One strategy to deliver microRNAs is to genetically engineer target cells to express microRNAs of interest. However, to control dosage and timing, as well as to limit potential side-effects, microRNAs' expression should ideally be under exogenous, inducible control. Conditional expression of miRNA-based short hairpin RNAs (shRNAmirs) via gene regulatory circuits such as the Tet-system is therefore a promising strategy to control shRNAmirs' expression in research and therapy. Single vector approaches like Tet-On all-in-one designs are more compatible with potential clinical applications by providing the Tet-On system components in a single round of genetic engineering. However, all-in-one systems often come at the expense of heterogeneous and unstable expression. In this study, we aimed to understand the causes that lead to such erratic transgene expression. By using a reporter cell, we found that the degree of heterogeneity mostly correlated with reverse tetracycline transactivator (rtTA) expression levels. Moreover, the targeted integration of a potent rtTA expression cassette into a genomic safe harbor locus functionally rescued previously silenced rtTA-responsive transcription units. Overall, our results suggest that ensuring homogenous and stable rtTA expression is essential for the robust and reliable performance of future Tet-On all-in-one designs.

Impact of procedural variability and study design quality on the efficacy of cell-based therapies for heart failure - a meta-analysis.

BACKGROUND: Cell-based therapy has long been considered a promising strategy for the treatment of heart failure (HF). However, its effectiveness in the clinical setting is now doubted. Because previous meta-analyses provided conflicting results, we sought to review all available data focusing on cell type and trial design. METHODS AND FINDINGS: The electronic databases PubMed, Cochrane library, ClinicalTrials.gov, and EudraCT were searched for randomized controlled trials (RCTs) utilizing cell therapy for HF patients from January 1, 2000 to December 31, 2020. Forty-three RCTs with 2855 participants were identified. The quality of the reported study design was assessed by evaluating the risk-of-bias (ROB). Primary outcomes were defined as mortality rate and left ventricular ejection fraction (LVEF) change from baseline. Secondary outcomes included both heart function data and clinical symptoms/events. Between-study heterogeneity was assessed using the I2 index. Subgroup analysis was performed based on HF type, cell source, cell origin, cell type, cell processing, type of surgical intervention, cell delivery routes, cell dose, and follow-up duration. Only 10 of the 43 studies had a low ROB for all method- and outcome parameters. A higher ROB was associated with a greater increase in LVEF. Overall, there was no impact on mortality for up to 12 months follow-up, and a clinically irrelevant average LVEF increase by LVEF (2.4%, 95% CI = 0.75-4.05, p = 0.004). Freshly isolated, primary cells tended to produce better outcomes than cultured cell products, but there was no clear impact of the cell source tissue, bone marrow cell phenotype or cell chricdose (raw or normalized for CD34+ cells). A meaningful increase in LVEF was only observed when cell therapy was combined with myocardial revascularization. CONCLUSIONS: The published results suggest a small increase in LVEF following cell therapy for heart failure, but publication bias and methodologic shortcomings need to be taken into account. Given that cardiac cell therapy has now been pursued for 20 years without real progress, further efforts should not be made. STUDY REGISTRY NUMBER: This meta-analysis is registered at the international prospective register of systematic reviews, number CRD42019118872.

Pathology and Advanced Imaging-Characterization of a Congenital Cardiac Defect and Complex Hemodynamics in a Pig: A Case Report.

Domestic pigs are widely used in cardiovascular research as the porcine circulatory system bears a remarkable resemblance to that of humans. In order to reduce variability, only clinically healthy animals enter the study as their health status is assessed in entry examination. Like humans, pigs can also suffer from congenital heart disease, such as an atrial septal defect (ASD), which often remains undetected. Due to the malformation of the endocardial cushion during organ development, mitral valve defects (e.g., mitral clefts) are sometimes associated with ASDs, further contributing to hemodynamic instability. In this work, we report an incidental finding of a hemodynamically highly relevant ASD in the presence of incompetent mitral and tricuspid valves, in an asymptomatic, otherwise healthy juvenile pig. In-depth characterization of the cardiac blood flow by four-dimensional (4D) flow magnetic resonance imaging (MRI) revealed a prominent diastolic left-to-right and discrete systolic right-to-left shunt, resulting in a pulmonary-to-systemic flow ratio of 1.8. Severe mitral (15 mL/stroke) and tricuspid (22 mL/stroke) regurgitation further reduced cardiac output. Pathological examination confirmed the presence of an ostium primum ASD and found a serous cyst of lymphatic origin that was filled with clear fluid partially occluding the ASD. A large mitral cleft was identified as the most likely cause of severe regurgitation, and histology showed mild to moderate endocardiosis in the coaptation area of both atrio-ventricular valves. In summary, although not common, congenital heart defects could play a role as a cause of experimental variability or even intra-experimental mortality when working with apparently heathy, juvenile pigs.

Hopes and Hurdles of Employing Mesenchymal Stromal Cells in the Treatment of Cardiac Fibrosis.

Excessive cardiac fibrosis plays a crucial role in almost all types of heart disease. Generally, cardiac fibrosis is a scarring process triggered in response to stress, injury, or aging and is characterized by the accumulation of activated myofibroblasts that deposit high levels of extracellular matrix proteins in the myocardium. While it is beneficial for cardiac repair in the short term, it can also result in pathological remodeling, tissue stiffening, and cardiac dysfunction, contributing to the progression of heart failure, arrhythmia, and sudden cardiac death. Despite its high prevalence, there is a lack of effective and safe therapies that specifically target myofibroblasts to inhibit or even reverse pathological cardiac fibrosis. In the past few decades, cell therapy has been under continuous evaluation as a potential treatment strategy, and several studies have shown that transplantation of mesenchymal stromal cells (MSCs) can reduce cardiac fibrosis and improve heart function. Mechanistically, it is believed that the heart benefits from MSC therapy by stimulating innate anti-fibrotic and regenerative reactions. The mechanisms of action include paracrine signaling and cell-to-cell interactions. In this review, we provide an overview of the anti-fibrotic properties of MSCs and approaches to enhance them and discuss future directions of MSCs for the treatment of cardiac fibrosis.

Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices.

BACKGROUND: Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. METHODS: LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. RESULTS: Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. CONCLUSIONS: We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases.

MiRNA Profiles of Extracellular Vesicles Secreted by Mesenchymal Stromal Cells-Can They Predict Potential Off-Target Effects?

The cardioprotective properties of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are currently being investigated in preclinical studies. Although microRNAs (miRNAs) encapsulated in EVs have been identified as one component responsible for the cardioprotective effect of MSCs, their potential off-target effects have not been sufficiently characterized. In the present study, we aimed to investigate the miRNA profile of EVs isolated from MSCs that were derived from cord blood (CB) and adipose tissue (AT). The identified miRNAs were then compared to known targets from the literature to discover possible adverse effects prior to clinical use. Our data show that while many cardioprotective miRNAs such as miR-22-3p, miR-26a-5p, miR-29c-3p, and miR-125b-5p were present in CB- and AT-MSC-derived EVs, a large number of known oncogenic and tumor suppressor miRNAs such as miR-16-5p, miR-23a-3p, and miR-191-5p were also detected. These findings highlight the importance of quality assessment for therapeutically applied EV preparations.

Treatment With Hydrolyzed Diet Supplemented With Prebiotics and Glycosaminoglycans Alters Lipid Metabolism in Canine Inflammatory Bowel Disease.

Canine inflammatory bowel disease (IBD) is a chronic, immunologically mediated intestinal disorder, resulting from the complex interaction of genetic, environmental and immune factors. Hydrolyzed diets are used in dogs with food-responsive diarrhea (FRD) to reduce adverse responses to immunostimulatory proteins. Prebiotics (PRBs) and glycosaminoglycans (GAGs) have previously been demonstrated to show anti-inflammatory activity in the intestinal mucosa. Notably, hydrolyzed diets combined with the administration of PRBs and GAGs offer a promising approach for the treatment of canine IBD. Our aim was to investigate the effects of hydrolyzed diet and GAG+PRB co-treatment on the serum metabolomic profile of IBD dogs. Dogs with IBD randomly received either hydrolyzed diet supplemented with GAGs and PRBs (treatment 1) or hydrolyzed diet alone (treatment 2) for 10 weeks. A targeted metabolomics approach using mass spectrometry was performed to quantify changes in the serum metabolome before and after treatment and between treatment 1 and 2. Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA) and univariate statistics were used to identify differences between the treatment groups. PCA, PLS-DA, and HCA showed a clear clustering of IBD dogs before and after hydrolyzed diet, indicating that the treatment impacted the serum metabolome. Univariate analysis revealed that most of the altered metabolites were involved in lipid metabolism. The most impacted lipid classes were components of cell membranes, including glycerophospholipids, sphingolipids, and di- and triglycerides. In addition, changes in serum metabolites after GAG+PRB co-treatment suggested a possible additional beneficial effect on the lipid metabolism in IBD dogs. In conclusion, the present study showed a significant increase in metabolites that protect gut cell membrane integrity in response to hydrolyzed diet alone or in combination with GAG+PRB co-treatment. Administration of such treatment over 70 days improved selected serum biomarkers of canine IBD, possibly indicating improved intestinal membrane integrity.

Human mesenchymal stromal cells and derived extracellular vesicles: Translational strategies to increase their proangiogenic potential for the treatment of cardiovascular disease.

Mesenchymal stromal cells (MSCs) offer great potential for the treatment of cardiovascular diseases (CVDs) such as myocardial infarction and heart failure. Studies have revealed that the efficacy of MSCs is mainly attributed to their capacity to secrete numerous trophic factors that promote angiogenesis, inhibit apoptosis, and modulate the immune response. There is growing evidence that MSC-derived extracellular vesicles (EVs) containing a cargo of lipids, proteins, metabolites, and RNAs play a key role in this paracrine mechanism. In particular, encapsulated microRNAs have been identified as important positive regulators of angiogenesis in pathological settings of insufficient blood supply to the heart, thus opening a new path for the treatment of CVD. In the present review, we discuss the current knowledge related to the proangiogenic potential of MSCs and MSC-derived EVs as well as methods to enhance their biological activities for improved cardiac tissue repair. Increasing our understanding of mechanisms supporting angiogenesis will help optimize future approaches to CVD intervention.

Effects of Long-Term Storage at -80 °C on the Human Plasma Metabolome.

High-quality biological samples are required for the favorable outcome of research studies, and valid data sets are crucial for successful biomarker identification. Prolonged storage of biospecimens may have an artificial effect on compound levels. In order to investigate the potential effects of long-term storage on the metabolome, human ethylenediaminetetraacetic acid (EDTA) plasma samples stored for up to 16 years were analyzed by gas and liquid chromatography-tandem mass spectrometry-based metabolomics. Only 2% of 231 tested plasma metabolites were altered in the first seven years of storage. However, upon longer storage periods of up to 16 years and more time differences of few years significantly affected up to 26% of the investigated metabolites when analyzed within subject age groups. Ontology classes that were most affected included complex lipids, fatty acids, energy metabolism molecules, and amino acids. In conclusion, the human plasma metabolome is adequately stable to long-term storage at -80 °C for up to seven years but significant changes occur upon longer storage. However, other biospecimens may display different sensitivities to long-term storage. Therefore, in retrospective studies on EDTA plasma samples, analysis is best performed within the first seven years of storage.

Shrunken pore syndrome, preeclampsia, and markers of NO metabolism in pregnant women during the first trimester.

Shrunken pore syndrome (SPS) is a condition that manifests itself as the decreased renal clearance of low-molecular-weight proteins but normal clearance of creatinine. Pregnant women with evidence of SPS during the first trimester have an increased risk of developing preeclampsia (PE). The nitric oxide (NO) metabolism markers arginine and ADMA, especially their ratio (Arg/ADMA), are recognized markers of endothelial dysfunction. The aim of this nested case-control study was to establish first-trimester reference intervals (RI) for markers of NO metabolism and to study these markers in women with evidence of SPS at the end of the first trimester. Seventy-four women were stratified in the first trimester according to evidence of SPS (SPS + or SPS-) and the occurrence of PE during subsequent pregnancy (PE + or PE-), as follows: SPS-/PE-, SPS+/PE-, SPS-/PE+, and SPS+/PE+. RIs were determined according to the CLSI EP28-A3c guidelines. Serum Arg and ADMA levels were analyzed. The Arg and ADMA concentrations did not differ among the four groups. However, women in the SPS+/PE + group had a significantly lower Arg/ADMA ratio than those in the other 3 groups (p = .02). In conclusion, we defined the first-trimester RI of Arg, ADMA and the Arg/ADMA ratio as markers of NO metabolism. Our results suggest that SPS in the first trimester predicts a pathophysiological hallmark of subsequent PE, i.e. lower NO production leading to increased vessel tone. Early identification of women at risk for later PE could lead to adaptive prophylactic interventions, such as supplementation with Arg or an NO-donor drug in order to mitigate the risk of developing PE.

Impact of Prolonged Blood Incubation and Extended Serum Storage at Room Temperature on the Human Serum Metabolome.

Metabolomics is a powerful technology with broad applications in life science that, like other -omics approaches, requires high-quality samples to achieve reliable results and ensure reproducibility. Therefore, along with quality assurance, methods to assess sample quality regarding pre-analytical confounders are urgently needed. In this study, we analyzed the response of the human serum metabolome to pre-analytical variations comprising prolonged blood incubation and extended serum storage at room temperature by using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) -based metabolomics. We found that the prolonged incubation of blood results in a statistically significant 20% increase and 4% decrease of 225 tested serum metabolites. Extended serum storage affected 21% of the analyzed metabolites (14% increased, 7% decreased). Amino acids and nucleobases showed the highest percentage of changed metabolites in both confounding conditions, whereas lipids were remarkably stable. Interestingly, the amounts of taurine and O-phosphoethanolamine, which have both been discussed as biomarkers for various diseases, were 1.8- and 2.9-fold increased after 6 h of blood incubation. Since we found that both are more stable in ethylenediaminetetraacetic acid (EDTA) blood, EDTA plasma should be the preferred metabolomics matrix.