ABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, has caused nearly 7 million deaths worldwide. Severe cases are marked by an aggressive inflammatory response known as hypercytokinemia, contributing to endothelial damage. Although vaccination has reduced hospitalizations, hypercytokinemia persists in breakthrough infections, emphasizing the need for disease models mimicking this response. Using a 3D microphysiological system (MPS), we explored the vascular role in SARS-CoV-2-induced hypercytokinemia. Methods: The vascularized micro-organ (VMO) MPS, consisting of human-derived primary endothelial cells (ECs) and stromal cells within an extracellular matrix, was used to model SARS-CoV-2 infection. A non-replicative pseudotyped virus fused to GFP was employed, allowing visualization of viral entry into human ECs under physiologic flow conditions. Expression of ACE2, TMPRSS2, and AGTR1 was analyzed, and the impact of viral infection on ACE2 expression, vascular inflammation, and vascular morphology was assessed. Results: The VMO platform facilitated the study of COVID-19 vasculature infection, revealing that ACE2 expression increased significantly in direct response to shear stress, thereby enhancing susceptibility to infection by pseudotyped SARS-CoV-2. Infected ECs secreted pro-inflammatory cytokines, including IL-6 along with coagulation factors. Cytokines released by infected cells were able to activate downstream, non-infected EC, providing an amplification mechanism for inflammation and coagulopathy. Discussion: Our findings highlight the crucial role of vasculature in COVID-19 pathogenesis, emphasizing the significance of flow-induced ACE2 expression and subsequent inflammatory responses. The VMO provides a valuable tool for studying SARS-CoV-2 infection dynamics and evaluating potential therapeutics.
ABSTRACT
BACKGROUND: Left ventricular noncompaction (LVNC) is the third most common pediatric cardiomyopathy characterized by a thinned myocardium and prominent trabeculations. Next-generation genetic testing has led to a rapid increase in the number of genes reported to be associated with LVNC, but we still have little understanding of its pathogenesis. We sought to grade the strength of the gene-disease relationship for all genes reported to be associated with LVNC and identify molecular pathways that could be implicated. METHODS: Following a systematic PubMed review, all genes identified with LVNC were graded using a validated, semi-quantitative system based on all published genetic and experimental evidence created by the Clinical Genome Resource (ClinGen). Genetic pathway analysis identified molecular processes and pathways associated with LVNC. RESULTS: We identified 189 genes associated with LVNC: 11 (6%) were classified as definitive, 21 (11%) were classified as moderate, and 140 (74%) were classified as limited, but 17 (9%) were classified as no evidence. Of the 32 genes classified as definitive or moderate, the most common gene functions were sarcomere function (n=11; 34%), transcriptional/translational regulator (n=6; 19%), mitochondrial function (n=3; 9%), and cytoskeletal protein (n=3; 9%). Furthermore, 18 (56%) genes were implicated in noncardiac syndromic presentations. Lastly, 3 genetic pathways (cardiomyocyte differentiation via BMP receptors, factors promoting cardiogenesis in vertebrates, and Notch signaling) were found to be unique to LVNC and not overlap with pathways identified in dilated cardiomyopathy and hypertrophic cardiomyopathy. CONCLUSIONS: LVNC is a genetically heterogeneous cardiomyopathy. Distinct from dilated or hypertrophic cardiomyopathies, LVNC appears to arise from abnormal developmental processes.
Subject(s)
Cardiomyopathies , Cardiomyopathy, Dilated , Isolated Noncompaction of the Ventricular Myocardium , Animals , Cardiomyopathies/genetics , Child , Humans , Isolated Noncompaction of the Ventricular Myocardium/genetics , Phenotype , SarcomeresABSTRACT
The o-aminophenol-N,N,O-triacetic acid (APTRA) chelator is employed extensively as a metal-recognition moiety in fluorescent indicators for biological free Mg(2+), as well as in low-affinity indicators for the detection of high levels of cellular Ca(2+). Despite its widespread use in sensor design, the limited metal selectivity of this chelating moiety can lead to binding of competing cations that complicate the fluorescence-based detection of metals of interest in complex samples. Reported herein are the structural characterization of APTRA complexes with various biologically relevant cations, and the thermodynamic analysis of complex formation with Mg(2+), Ca(2+) and Zn(2+). Our results indicate that the low affinity of APTRA for Mg(2+), which makes it a suitable metal-recognition moiety for sensitive analysis of typical millimolar levels of this metal in cells, stems from a much higher enthalpic cost of Mg(2+) binding compared to that of other cations. The results are discussed in the context of indicator design, highlighting the aspects that may aid the future development of fluorescent sensors with enhanced metal selectivity profiles.
ABSTRACT
INTRODUCTION: Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes. METHODS: We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification. RESULTS: From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3'-untranslated regions (3'UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3'UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1. CONCLUSIONS: These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.