Citations

Introduction: Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease characterized by the underdeveloped left ventricle. Early serial postmortem examinations revealed a high rate of coronary artery abnormalities in HLHS fetal hearts (e.g., thickened wall and kinking arteries). However, the intrinsic defect in HLHS coronary vessels and its genetic basis remain unclear. Methods: We profiled human fetal heart with an underdeveloped left ventricle (ULV) and induced pluripotent stem cells derived endothelial cells (iPSC-ECs) from HLHS patients at single-cell resolution. CD144 + / NPR3 - vascular ECs were selected and classified as venous, arterial, and late arterial populations. To study the arterial EC phenotypes, we generated iPSC-arterial ECs (AECs, CDH5 + CXCR4 + NT5E -/low ) derived from 3 HLHS patients and 3 age-matched controls, and evaluated their functionalities including cell cycle regulation, angiogenesis, and inflammatory response. Results: Revealed by single cell RNA-seq and subsequent gene ontology analysis, ULV late arterial EC population showed significant defects in EC development, proliferation, angiogenesis, and Notch signaling compared to the control. Consistently, HLHS iPSCs exhibited impaired AEC differentiation judged by the reduced CXCR4 + NT5E -/low AEC progenitors. Mature HLHS iPSC-AECs showed reduced angiogenesis and enhanced G0/G1 cell cycle arrest with downregulated cell cycle-related genes (e.g., Ki67, CCND1/2 ). Healthy human aortic smooth muscle cells exhibited abnormal proliferation and synthetic phenotypes when co-cultured with HLHS iPSC-AECs. Additionally, NOTCH pathway genes (e.g., DLL4, HEY1, GJA5 ) were suppressed in both ULV AECs and HLHS iPSC-AECs. HLHS de novo variant KMT2D directly regulated the transcription of NOTCH targeted genes involved in arterial development and proliferation via H3K4me2. Intriguingly, the treatment of NOTCH ligands (Jag1, Dll1) significantly improved the proliferation of HLHS AECs. Conclusions: Our study revealed that HLHS coronary AECs were dysfunctional in angiogenesis, proliferation, and EC-SMC interaction. KMT2D-NOTCH signaling may contribute to the impaired development and proliferation of HLHS AECs.

Synaptic loss is the structural basis for memory impairment in Alzheimer’s disease (AD). While the underlying pathological mechanism remains elusive, it is known that misfolded proteins accumulate as β-amyloid (Aβ) plaques and hyperphosphorylated Tau tangles decades before the onset of clinical disease. The loss of Pin1 facilitates the formation of these misfolded proteins in AD. Pin1 protein controls cell-cycle progression and determines the fate of proteins by the ubiquitin proteasome system. The activity of the ubiquitin proteasome system directly affects the functional and structural plasticity of the synapse. We localized Pin1 to dendritic rafts and postsynaptic density (PSD) and found the pathological loss of Pin1 within the synapses of AD brain cortical tissues. The loss of Pin1 activity may alter the ubiquitin-regulated modification of PSD proteins and decrease levels of Shank protein, resulting in aberrant synaptic structure. The loss of Pin1 activity, induced by oxidative stress, may also render neurons more susceptible to the toxicity of oligomers of Aβ and to excitation, thereby inhibiting NMDA receptor-mediated synaptic plasticity and exacerbating NMDA receptor-mediated synaptic degeneration. These results suggest that loss of Pin1 activity could lead to the loss of synaptic plasticity in the development of AD.

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA‐containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in‐person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.