Medicine

Following myocardial infarction (MI), myocardial cells undergo cell death, and the necrotic region is replaced by extracellular matrix (ECM) proteins such as collagens. Myofibroblasts are responsible for producing these ECM proteins. Cardiac myofibroblasts are differentiated from resident fibroblasts in response to inflammation. To date, genetically modified mice driven by the Periostin promoter and adeno-associated virus 9 (AAV9) carrying the Periostin promoter have been used for gene transfer into cardiac myofibroblasts. However, these methods require multiple steps and are time-consuming and expensive. Therefore, we developed a method for delivering genes into cardiac myofibroblasts using retroviruses. Specifically, the DNA of the target gene was transfected into Plat-E cells, which are packaging cells, to generate retroviruses. The virus-containing supernatant was then harvested, and the viruses were pelleted by centrifugation and suspended in PBS-containing polybrene. Subsequently, permanent occlusion of the left coronary artery was performed, and 20 μL of viral solution was immediately administered using a 29G needle at a position 1–2 mm below the ligation site in the heart of mice maintained in an open chest state. Using this method, we were able to introduce genes into the myofibroblasts of interest surrounding the MI site.

Cardiovascular disease, the current leading cause of death worldwide, is a multifactorial disorder that involves a strong contribution of both the innate and adaptive immune systems. Overactivation of the immune system and inappropriate secretion of pro-inflammatory cytokines lead to vascular impairments and the development of cardiovascular disorders, including hypertension, atherosclerosis, and peripheral artery disease. Lymphocytes, macrophages, and dendritic cells can all secrete pro-inflammatory cytokines. This makes it challenging to isolate a specific subset of immune cells, particularly cytokines, and their contribution to vascular dysfunction remains difficult to elucidate. To solve this problem, our laboratory has developed the novel “immune cell-aorta” co-culture system described herein. This experimental protocol enables investigators to isolate an immune cell of interest and identify the cytokine(s) at the origin of vascular alterations.

Assessing thrombogenicity is crucial for evaluating biomaterials, especially those that interface with flowing blood, such as cardiovascular implants, including vascular stents, grafts, and stent-grafts. To standardize thrombogenicity assessments, we use human plasma and quantify the light absorbance associated with the biomaterial. For this evaluation, various tubular vascular implants from leading brands—such as bare-metal stents, drug-eluting stents, vascular grafts, and stent-grafts—are longitudinally sectioned into small pieces and placed in a low-adhesion 96-well plate. Using either platelet-rich plasma (PRP) or platelet-poor plasma (PPP), we measure the absorbance of light passing through the plate over an hour and plot the resulting curve. This method quantifies the thrombogenicity of a biomaterial under controlled conditions. Key factors examined include anticoagulants, platelet presence, and surface-coating molecules to assess their impact on thrombogenicity. Using this simple, reproducible protocol, we demonstrated (a) the relative efficacy of various anticoagulants in thrombogenicity assessments, and (b) the effectiveness of vascular coating molecules in reducing thrombogenicity on stents. This streamlined approach offers valuable insights for optimizing biomaterial performance in vascular implants. Unlike conventional clotting assays, which focus on standardized blood clotting mechanisms, this assay is tailored to evaluate biomaterials and external parameters influencing thrombogenicity.

Accurate quantification of von Willebrand factor ristocetin cofactor activity (VWF:RCo) is critical for the diagnosis and classification of von Willebrand disease, the most common hereditary and acquired bleeding disorder in humans. Moreover, it is important to accurately assess the function of von Willebrand factor (VWF) concentrates within the pharmaceutical industry to provide consistent and high-quality biopharmaceuticals. Although the performance of VWF:RCo assay has been improved by using coagulation analyzers, which are specialized devices for blood and blood plasma samples, scientists still report a high degree of intra- and inter-assay variation in clinical laboratories. Moreover, high, manual sample dilutions are required for VWF:RCo determination of VWF concentrates within the pharmaceutical industry, which are a major source for assay imprecision. For the first time, we present a precise and accurate method to determine VWF:RCo, where all critical pipetting and mixing steps are automated. A pre-dilution setup was established on CyBio FeliX (Analytik-Jena) liquid handling system, and an adapted VWF:RCo method on BCS-XP analyzer (Siemens) is used. The automated pre-dilution method was executed on three different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. Comparative sample testing revealed a similar assay precision (coefficient of variation = 5.9% automated, 3.1% manual pre-dilution) and no significant differences between the automated approach and manual dilutions of an expert in this method. While no outliers were generated with the automated procedure, the manual pre-dilution resulted in an error rate of 8.3%. Overall, this operator-independent protocol enables standardization and offers an efficient way of fully automating VWF activity assays, while maintaining the precision and accuracy of an expert analyst.

Current ischemic models strive to replicate ischemia-mediated injury. However, they face challenges such as inadequate reproducibility, difficulties in translating rodent findings to humans, and ethical, financial, and practical constraints that limit the accuracy of extensive research. This study introduces a novel approach to inducing persistent ischemia in 3-day-old chicken embryos using endothelin-1. The protocol targets the right vitelline arteries, validated with Doppler blood flow imaging and molecular biology experiments. This innovative approach facilitates the exploration of oxidative stress, inflammatory responses, cellular death, and potential drug screening suitability utilizing a 3-day-old chicken embryo.

Apolipoprotein B (APOB) is the primary structural protein of atherogenic lipoproteins, which drive atherogenesis and thereby lead to deadly cardiovascular diseases (CVDs). Plasma levels of APOB-containing lipoproteins are tightly modulated by LDL receptor–mediated endocytic trafficking and cargo receptor–initiated exocytic route; the latter is much less well understood. This protocol aims to present an uncomplicated yet effective method for detecting APOB/lipoprotein secretion. We perform primary mouse hepatocyte isolation and culture coupled with well-established techniques such as immunoblotting for highly sensitive, specific, and semi-quantitative analysis of the lipoprotein secretion process. Its inherent simplicity facilitates ease of operation, rendering it a valuable tool widely utilized to explore the intricate landscape of cellular lipid metabolism and unravel the mechanistic complexities underlying lipoprotein-related diseases.

Pulmonary hypertension (PH) is a group of pulmonary vascular disorders in which mean pulmonary arterial pressure (mPAP) becomes abnormally high because of various pathological conditions, including remodeling of the pulmonary arteries, lung and heart disorders, or congenital conditions. Various animal models, including mouse and rat models, have been used to recapitulate elevated mPAP observed in PH patients. However, the measurement and recording of mPAP and mean systemic arterial pressure (mSAP) in small animals require microsurgical procedures and a sophisticated data acquisition system. In this paper, we describe the surgical procedures for right heart catheterizations (RHC) to measure mPAP in rats. We also explain the catheterization of the carotid artery for simultaneous measurement of mPAP and mSAP using the PowerLab Data Acquisition system. We enumerate the surgical steps involved in exposing the jugular vein and the carotid artery for catheterizing these two blood vessels. We list the tools used for microsurgery in rats, describe the methods for preparing catheters, and illustrate the process for inserting the catheters in the pulmonary and carotid arteries. Finally, we delineate the steps involved in the calibration and setup of the PowerLab system for recording both mPAP and mSAP. This is the first protocol wherein we meticulously explain the surgical procedures for RHC in rats and the recording of mPAP and mSAP. We believe this protocol will be essential for PH research. Investigators with little training in animal handling can reproduce this microsurgical procedure for RHC in rats and measure mPAP and mSAP in rat models of PH. Further, this protocol is likely to help master RHC in rats that are performed for other conditions, such as heart failure, congenital heart disease, heart valve disorders, and heart transplantation.

Cardiovascular diseases are the leading cause of death and morbidity worldwide. Patient mortality has been successfully reduced by nearly half in the last four decades, mainly due to advances in minimally invasive surgery techniques and interventional cardiology methods. However, a major hurdle is still the translational gap between preclinical findings and the conversion into effective therapies, which is partly due to the use of model systems that fail to recapitulate key aspects of human physiology and disease. Large animal models such as pigs and non-human primates are highly valuable because they closely resemble humans but are costly and time intensive. Here, we provide a method for long-term ex vivo culture of non-human primate (NHP) myocardial tissue that offers a powerful alternative for a wide range of applications including electrophysiology studies, drug screening, and gene function analyses.

Graphical overview

Genome-wide CRISPR-based screening is a powerful tool in forward genetics, enabling biologic discovery by linking a desired phenotype to a specific genetic perturbation. However, hits from a genome-wide screen require individual validation to reproduce and accurately quantify their effects outside of a pooled experiment. Here, we describe a step-by-step protocol to rapidly assess the effects of individual sgRNAs from CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. All steps, including cloning, lentivirus generation, cell transduction, and phenotypic readout, can be performed entirely in 96-well plates. The system is highly flexible in both cell type and selection system, requiring only that the phenotype(s) of interest be read out via flow cytometry. We expect that this protocol will provide researchers with a rapid way to sift through potential screening hits, and prioritize them for deeper analysis in more complex in vitro or even in vivo systems.

Graphical abstract

Atherosclerosis, a condition characterized by thickening of the arteries due to lipid deposition, is the major contributor to and hallmark of cardiovascular disease. Although great progress has been made in lowering the lipid plaques in patients, the conventional therapies fail to address the needs of those that are intolerant or non-responsive to the treatment. Therefore, additional novel therapeutic approaches are warranted. We have previously shown that increasing the cellular amounts of microRNA-30c (miR-30c) with the aid of viral vectors or liposomes can successfully reduce plasma cholesterol and atherosclerosis in mice. To avoid the use of viruses and liposomes, we have developed new methods to synthesize novel miR-30c analogs with increasing potency and efficacy, including 2’-O-methyl (2’OMe), 2’-fluoro (2’F), pseudouridine (ᴪ), phosphorothioate (PS), and N-acetylgalactosamine (GalNAc). The discovery of these modifications has profoundly impacted the modern RNA therapeutics, as evidenced by their increased nuclease stability and reduction in immune responses. We show that modifications on the passenger strand of miR-30c not only stabilize the duplex but also aid in a more readily uptake by the cells without the aid of viral vectors or lipid emulsions. After uptake, the analogs with PS linkages and GalNAc-modified ribonucleotides significantly reduce the secretion of apolipoprotein B (ApoB) without affecting apolipoprotein A1 (ApoA1) in human hepatoma Huh-7 cells. We envision an enormous potential for these modified miR-30c analogs in therapeutic intervention for treating cardiovascular diseases.