Hyperoxia impairs induced pluripotent stem cell-derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype.

Background: Induced pluripotent stem cells (iPSCs) directed to endothelial identity (iPSC-ECs) are emerging as a potent tool for regenerative medicine in vascular disease. However, iPSC-ECs lose expression of key identity markers under standard in vitro conditions, limiting their clinical applications. Methods: To model physiological in vivo conditions, we examined the bioenergetics, presence of key cell markers, and proliferative and angiogenic capacity in iPSC-ECs at late and early passage under hyperoxic (21%) and physiological (4%) oxygen concentrations. Results: Physoxia resulted in relative preservation of mitochondrial bioenergetic activity, as well as CD144 expression in late passage iPSC-ECs, but not proliferative capacity or tube formation. Single cell RNA sequencing (scRNA-seq) revealed that late passage hyperoxic iPSC-ECs develop an endothelial-to-mesenchymal phenotype. Comparing scRNA-seq data from iPSC-ECs and from atherosclerotic ECs revealed overlap of their transcriptional phenotypes. Conclusions: Taken together, our studies demonstrate that physiological 4% oxygen culture conditions were sufficient to improve mitochondrial function in high passage cells, but alone was insufficient to preserve angiogenic capacity. Furthermore, late passage cells under typical conditions take on an endothelial-to-mesenchymal phenotype with similarities to ECs found in atherosclerosis.

A Biomimetic Leaflet Scaffold for Aortic Valve Remodeling.

Heart valve disease poses a significant clinical challenge, especially in pediatric populations, due to the inability of existing valve replacements to grow or respond biologically to their microenvironment. Tissue-engineered heart valves (TEHVs) provide a solution by facilitating patient-specific models for self-repair and remodeling. In this study, a 3D-bioprinted TEHV is designed to emulate the trilayer leaflet structure of an aortic valve. A cell-laden hydrogel scaffold made from gelatin methacrylate and polyethylene glycol diacrylate (GelMA/PEGDA) incorporates valvular interstitial-like (VIC-like) cells, being reinforced with a layer of polycaprolactone (PCL). The composition of the hydrogel scaffold remains stable over 7 days, having increased mechanical strength compared to pure GelMA. The scaffold maintains VIC-like cell function and promotes extracellular matrix (ECM) protein expression up to 14 days under two dynamic culture conditions: shear stress and stretching; replicating heart valve behavior within a more physiological-like setting and suggesting remodeling potential via ECM synthesis. This TEHV offers a promising avenue for valve replacements, closely replicating the structural and functional attributes of a native aortic valve, leading to mechanical and biological integration through biomaterial-cellular interactions.

Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204.

Redox processes can modulate vascular pathophysiology. The endoplasmic reticulum redox chaperone protein disulfide isomerase A1 (PDIA1) is overexpressed during vascular proliferative diseases, regulating thrombus formation, endoplasmic reticulum stress adaptation, and structural remodeling. However, both protective and deleterious vascular effects have been reported for PDIA1, depending on the cell type and underlying vascular condition. Further understanding of this question is hampered by the poorly studied mechanisms underlying PDIA1 expression regulation. Here, we showed that PDIA1 mRNA and protein levels were upregulated (average 5-fold) in the intima and media/adventitia following partial carotid ligation (PCL). Our search identified that miR-204-5p and miR-211-5p (miR-204/211), two broadly conserved miRNAs, share PDIA1 as a potential target. MiR-204/211 was downregulated in vascular layers following PCL. In isolated endothelial cells, gain-of-function experiments of miR-204 with miR mimic decreased PDIA1 mRNA while having negligible effects on markers of endothelial activation/stress response. Similar effects were observed in vascular smooth muscle cells (VSMCs). Furthermore, PDIA1 downregulation by miR-204 decreased levels of the VSMC contractile differentiation markers. In addition, PDIA1 overexpression prevented VSMC dedifferentiation by miR-204. Collectively, we report a new mechanism for PDIA1 regulation through miR-204 and identify its relevance in a model of vascular disease playing a role in VSMC differentiation. This mechanism may be regulated in distinct stages of atherosclerosis and provide a potential therapeutic target.

Atherosclerotic three-layer nanomatrix vascular sheets for high-throughput therapeutic evaluation.

In vitro atherosclerosis models are essential to evaluate therapeutics before in vivo and clinical studies, but significant limitations remain, such as the lack of three-layer vascular architecture and limited atherosclerotic features. Moreover, no scalable 3D atherosclerosis model is available for making high-throughput assays for therapeutic evaluation. Herein, we report an in vitro 3D three-layer nanomatrix vascular sheet with critical atherosclerosis multi-features (VSA), including endothelial dysfunction, monocyte recruitment, macrophages, extracellular matrix remodeling, smooth muscle cell phenotype transition, inflammatory cytokine secretion, foam cells, and calcification initiation. Notably, we present the creation of high-throughput functional assays with VSAs and the use of these assays for evaluating therapeutics for atherosclerosis treatment. The therapeutics include conventional drugs (statin and sirolimus), candidates for treating atherosclerosis (curcumin and colchicine), and potential gene therapy (miR-146a-loaded liposomes). The high efficiency and flexibility of the scalable VSA functional assays should facilitate drug discovery and development for atherosclerosis.

Simulated microgravity improves maturation of cardiomyocytes derived from human induced pluripotent stem cells.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels.

EMID2 is a novel biotherapeutic for aggressive cancers identified by in vivo screening.

BACKGROUND: New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. METHODS: We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. RESULTS: EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFß maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. CONCLUSION: This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients.

HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.