Biology subjects

Nickenig, G.

Publications and source records attributed to Nickenig, G..

3 recordsLinked to original sources

bioRxiv2025

Large extracellular vesicles derived from red blood cells in coronary artery disease patients with anemia promote endothelial dysfunction

Background and purposeEndothelial dysfunction (ED) is a hallmark of cardiovascular disease (CVD). We recently showed that anemia is associated with worsening of endothelial function after acute myocardial infarction (AMI). Extracellular vesicles (EVs) are efficient communicators between cells and can functionally contribute to different CVD, including, AMI. However, their specific role of EVs in stable coronary artery disease (CAD)-associated with anemia, particularly their contribution to ED, has not yet been investigated systematically. Experimental approachRed blood cell-derived EVs (REVs) and plasma-derived EVs (PLEVs) from all blood cells and endothelium were isolated from patients with stable CAD. The isolated large REVs and PLEVs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting. Uptake assays were performed by co-incubating with fluorescently-labeled REVs and PLEVs with human umbilical vein endothelial cells (ECs). Nitric oxide (NO) consumption ability of REVs was analyzed using a chemiluminescence detector (CLD). After co-incubation of aortic rings explanted from wild-type (WT) mice with REVs and PLEVs from anemic and non-anemic CAD patients, endothelial function was assessed using a wire myograph system. To investigate differences in the content of REVs and PLEVs between anemic and non-anemic CAD patients, proteomic analysis was performed. Key resultsDLS analysis showed that both REVs and PLEVs were within the size distribution range of 100-1000 nm. NTA analysis revealed increased release of REVs in anemic patients compared to non-anemic patients. Co-incubation of labeled REVs and PLEVs with ECs demonstrated their uptake by ECs in vitro which was similar between anemic patients compared to non-anemic patients. REVs from anemic patients showed increased NO consumption compared to those from non-anemic patients. Aortic rings co-incubated with REVs from anemic patients showed attenuated endothelial NO-dependent relaxation responses compared to non-anemic patients. Proteomics analysis of REVs from anemic patients revealed numerous differentially expressed proteins, including decreased abundance of antioxidant proteins such as catalase 1 (CAT1), superoxide dismutase 1 (SOD1) and increased oxidative stress-promoting myeloperoxidase (MPO). Co-incubation of ECs with REVs from anemic patients demonstrated increased ROS production. ConclusionAnemia is associated with increased release of REVs and enhanced NO consumption, which promotes ED. This is further exacerbated by an altered redox balance and increased ROS production, implicating therapeutic importance in anemic patients with CAD. Graphical AbstractAnemia is associated with an increased release of RBC-derived large extracellular vesicles (REVs), which are taken up by endothelial cells (ECs). Anemic REVs show enhanced nitric oxide (NO) consumption, contributing to NO dysregulation in ECs. Additionally, REVs carry various redox enzymes, including the oxidative stress-promoting enzyme myeloperoxidase (MPO), as well as antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). An imbalance in these redox enzymes leads to increased oxidative stress and endothelial nitric oxide synthase (eNOS) uncoupling, resulting in impaired NO-mediated relaxation responses and subsequent endothelial dysfunction (ED). O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/642191v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19d4cccorg.highwire.dtl.DTLVardef@1d1b3forg.highwire.dtl.DTLVardef@e7f144org.highwire.dtl.DTLVardef@190b007_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

Porcine and human aortic valve endothelial and interstitial cell isolation and characterization

BackgroundCalcific aortic valve stenosis is defined by pathological changes in the aortic valve and their predominant cell types: valvular interstitial (VICs) and endothelial cells (VECs). Understanding the cellular and molecular mechanisms of this disease is a prerequisite to identify potential pharmacological treatment strategies. In this study, we present a unique aortic valve cell isolation technique to acquire specific human and porcine cell populations and compared VICs and VECs of these species with each other for the first time. Methods and ResultsAortic valve cells were isolated from human explants from patients undergoing surgical aortic valve replacement or porcine valvular tissue. Pure VEC and VIC populations could be verified by gene expression analysis and immunofluorescence staining showing a highly significant upregulation of endothelial markers in VECs and mesenchymal markers in VICs, respectively. Further analysis and comparison of cells in in vitro experiments revealed that endothelial-to-mesenchymal transition could be induced in hVECs, leading to significant increase of mesenchymal markers. In vitro calcification experiments of VICs induced by osteogenic medium or pro-calcifying medium demonstrated a pronounced calcification marker expression and visible calcific deposition in Alizarin red staining in both species. ConclusionThis study aims to initiate a first step towards standardization of a reproducible isolation technique for pure human and porcine VEC and VIC populations. Comparison of human and porcine aortic valve cells demonstrated that porcine cells might serve as an alternative cellular model system, in settings, where human tissues are difficult to obtain. Statements and DeclarationsThe authors declare no relevant financial or non-financial interests to disclose.

cell biology↗

bioRxiv2022

Aortic valve disease augments vesicular microRNA-145-5p to regulate the calcification of valvular interstitial cells via cellular crosstalk

RationaleAortic valve stenosis (AVS) is a major contributor to cardiovascular death in the elderly population worldwide. MicroRNAs (miRNAs) are highly dysregulated in patients with AVS undergoing surgical aortic valve replacement (SAVR). However, miRNA-dependent mechanisms regulating inflammation and calcification or miRNA-mediated cell-cell crossstalk during the pathogenesis of AVS are still poorly understood. Here, we explored the role of extracellular vesicles (EV)-associated miR-145-5p, which we showed to be highly upregulated upon valvular calcification in AVS in mice and humans. MethodsHuman TaqMan miRNA arrays identified dysregulated miRNAs in aortic valve tissue explants from AVS patients compared to non-calcified valvular tissue explants of patients undergoing SAVR. Echocardiographic parameters were measured in association with the quantification of dysregulated miRNAs in a murine AVS model. In vitro calcification experiments were performed to explore the effects of EV-miR-145-5p on calcification and crosstalk in valvular cells. To dissect molecular miRNA signatures and their effect on signaling pathways, integrated OMICS analyses were performed. RNA sequencing (RNA-seq), high-throughput transcription factor (TF) and proteome arrays showed that a number of genes, miRNAs, TFs, and proteins are crucial for calcification and apoptosis, which are involved in the pathogenesis of AVS. ResultsAmong several miRNAs dysregulated in valve explants of AVS patients, miR-145-5p was the most highly gender-independently dysregulated miRNA (AUC, 0.780, p-value, 0.01). MiRNA arrays utilizing patient-derived- and murine aortic-stenosis samples demonstrated that the expression of miR-145-5p is significantly upregulated and correlates positively with cardiac function based on echocardiography. In vitro experiments confirmed that miR-145-5p is encapsulated into EVs and shuttled into valvular interstitial cells. Based on the integrated OMICs results, miR-145-5p interrelates with markers of inflammation, calcification, and apoptosis. In vitro calcification experiments demonstrated that miR-145-5p regulates the ALPL gene, a hallmark of calcification in vascular and valvular cells. EV-mediated shuttling of miR-145-5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3 untranslated region to inhibit its translation, thereby diminishing the calcification of target valvular interstitial cells. ConclusionElevated levels of pro-calcific and pro-apoptotic EV-associated miR-145-5p contribute to the progression of AVS via the ZEB2-ALPL axis, which could potentially be therapeutically targeted to minimize the burden of AVS. Clinical SignificanceO_ST_ABSWhat is known?C_ST_ABSO_LIAortic valve stenosis (AVS) is the most prevalent structural heart valve disease requiring surgical or interventional valve replacement. Currently, no medical treatment option is available to slow, halt, or reverse the progression of the disease. C_LIO_LIAVS induces pressure overload on the left ventricle (LV), resulting in concentric hypertrophy and LV dysfunction. C_LIO_LIAVS is not an exclusively degenerative disease that leads to fibrosis and calcification of the valve cusps but rather a chronic inflammatory disease, in which mechanical strain and shear stress lead to endothelial dysfunction and immune cell infiltration, which induces chronic inflammation, apoptosis and differentiation of valvular interstitial cells into osteoblast-like cells. C_LIO_LIIncreasing osteoblastic differentiation and the formation of macrocalcifications are hallmarks of the later stages of AVS. C_LI What is the new information we provide?O_LIDuring aortic valve stenosis, expression pattern of vesicle-associated regulatory miRNAs is altered. C_LIO_LIPatient-derived aortic valve tissue demonstrated an increased expression of miR-145-5p in humans, as well as in aortic valve explants from an experimental murine AVS model. C_LIO_LIMiR145-5p contributes to calcification of the aortic valve through ZEB2, a transcriptional repressor of ALPL, in valvular interstitial cells. C_LIO_LIExtracellular vesicular shuttling of miR-145-5p contributes to valvular cell-cell crosstalk and plays a role in the pathogenesis of AVS. C_LI

cell biology↗