Irina B. Beloglazova

Tissue engineered constructs generated in vitro are a promising tool for repairing damaged organs and tissues. Recent progress enables the fabrication of thick, physiologically relevant grafts that replicate native tissue structure. However, their clinical translation is limited by the lack of a functional vascular network, which is essential for construct formation and maturation in vitro, and rapid integration with the host vessels after transplantation. The aim of this study was to develop a method of prevascularized cell sheet fabrication based on a combination of cardiac mesenchymal stromal cells (cardiosphere-derived cells) and endothelial cells. Methods: Prevascularized cell sheets were formed by co-culturing murine cardiosphere-derived cells with endothelial cells at a 4 : 1 ratio. Microscopy methods were used to assess the size, thickness, viability and the formation of capillary-like structures. To visualize capillary-like structures, endothelial cells were pre-stained with CFDA SE. Cell viability was assessed using the LIVE/DEAD assay. We investigated the influence of different culture media (serum-free medium, medium containing 10% serum, and/or EGM-2 MV) and endothelial cell seeding strategies (mixing vs. seeding onto pre-plated cardiosphere-derived cells) on these parameters. Results: We developed the prevascularized cell sheet fabrication method based on combination of cardiosphere-derived and endothelial cells. Culture medium composition influenced cell sheet stability and prevascularization. EGM-2 MV caused spontaneous detachment of cell sheets, while both serum-free and serum-containing IMDM supported formation of stable constructs. However, serum-free medium reduced cell viability and resulted in fewer capillary-like structures compared to IMDM with 10% serum. The endothelial cell seeding strategy did not affect prevascularization, and mixing was adopted as technically simpler approach. Conclusion: Optimal conditions for prevascularized cell sheet fabrication include co-culturing cardiosphere-derived and endothelial cells in IMDM supplemented with 10% serum for three days. This method provides a foundation for generating prevascularized tissue constructs and represents an important step toward engineering clinically relevant vascularized tissues and organs.

Epicardial mesothelium plays a pivotal role in postinfarction cardiac repair by generating fibroblasts, producing extracellular matrix, and releasing paracrine mechanisms. However, interspecies differences have not been sufficiently studied, particularly in in vivo models of scar-free healing such as the African spiny mouse (Acomys cahirinus). This study aimed to compare the profibrotic response of epicardial mesothelial cells (MCs) from Acomys and C57BL/6 mice to hypoxic stress, a key factor in postinfarction recovery. We isolated epicardial MCs from the African spiny mouse (Acomys cahirinus), a species with documented cardiac regenerative capabilities, and from C57BL/6 laboratory mice. Using a CoCl₂-induced hypoxia model in vitro, we assessed cell viability, morphological changes, and expression of epithelial and fibroblast markers. In vivo, following experimental myocardial infarction (MI), we evaluated tissue hypoxia (pimonidazole adducts), epicardial activation (layer thickness, Wt1⁺ and TBX18⁺ progenitor cells), and collagen accumulation. The study was conducted using real-time PCR, Western blotting, immunohistochemical analysis and microscopic examination. In vitro, MCs from both species exhibited an epithelial-like phenotype under normoxic conditions, expressing E-cadherin and cytokeratin 18. Hypoxia (200 µM CoCl₂) induced a comparable response in both Acomys and C57BL/6 cells, characterized by a shift to a spindle-shaped, fibroblast-like morphology, decreased E-cadherin expression, and increased pro-collagen 1 and α-SMA expression. Following MI, both species exhibited similarly extensive hypoxic areas affecting the epicardial zone. Epicardial activation dynamics were comparable: from day 3 post-MI, epicardial thickness increased significantly, and Wt1⁺ and TBX18⁺ progenitor cells accumulated, peaking during the first week. Collagen accumulation in the epicardial region was similar between species, although the number of Wt1⁺ cells was higher in C57BL/6 on day 7. Despite the well-known superior regenerative capacity of spiny mice, epicardial MCs from Acomys and C57BL/6 demonstrated similar signs of profibrotic responses to hypoxic stimulation both in vitro and following MI. These findings suggest that species-specific regenerative outcomes may not be attributable to differential acute epicardial sensitivity to hypoxia, but rather to downstream mechanisms or additional factors influencing the cardiac repair process. This study provides the first characterization of Acomys epicardial MCs and establishes a foundation for further investigation of evolutionarily conserved and species-specific mechanisms of cardiac regeneration.

Background: Cardiac fibrosis represents a significant health burden, with endothelial dysfunction and damaged perivascular microenvironment increasingly recognized as key contributors to fibrotic remodeling. The urokinase plasminogen activator receptor (uPAR), a critical component of the urokinase system, plays a pivotal role in vascular remodeling and fibrosis. While prior evidence indicates that uPAR deficiency leads to microvascular dysfunction and perivascular fibrosis, the underlying mechanisms remain poorly defined. This study investigates how uPAR deficiency contributes to fibrotic remodeling of the cardiac perivascular-like microenvironment. Methods: Single-cell RNA sequencing data analysis and immunofluorescence staining on mouse heart cryosections were performed to characterize uPAR expression within the cardiac perivascular microenvironment. To model this microenvironment in vitro, cardiospheres (CSs) were generated from non-myocyte cardiac cells of wild-type and uPAR-knockout mice. CRISPR/Cas9-generated Plaur knockout (KO) 3T3 fibroblasts (FBs) were employed as model stromal cells. Pro-fibrotic activation of FBs was induced by TGFβ1 treatment. Comparative analyses of extracellular matrix (ECM) deposition, fibrotic cell transformation, and comprehensive secretome profiling was conducted using western blotting. Results: Our findings demonstrated that uPAR was expressed by endothelial cells (ECs) and FBs within the cardiac perivascular microenvironment. uPAR deficiency exacerbated profibrotic stimuli in CSs, including elevated active TGFβ1, impaired integrin functions, and altered cell secretome. These alterations collectively disrupt critical cell-cell and cell-matrix interactions, leading to increased ECM deposition, EC loss and decreased cell viability. Using Plaur KO FBs, we demonstrated that uPAR deficiency amplified TGFβ1-mediated Akt signaling pathway and ECM deposition. Conclusions: Our study reveals that uPAR loss drives fibrotic remodeling of the cardiac perivascular-like microenvironment and exacerbates TGFβ1-mediated effects, highlighting its potential as a therapeutic target for cardiac fibrosis.

Mesothelial cells line serosal cavities and internal organs, playing a vital role in maintaining serosal integrity and homeostasis. Their remarkable plasticity and ability to undergo mesothelial-to-mesenchymal transition (MMT) position them as key regulators of tissue repair. However, when normal repair processes fail, mesothelial cells can acquire a profibrotic phenotype. They actively contribute to all stages of fibrosis development, including inflammation, fibrin accumulation, myofibroblast differentiation, and extracellular matrix (ECM) remodeling. Fibrotic progression involves multiple cell types, and communication among them is essential for its perpetuation. Mesothelial cells are implicated in bidirectional crosstalk with fibroblasts, macrophages, lymphocytes, and endothelial cells of the serosal microenvironment through direct contact, paracrine signaling, and extracellular vesicle exchange. These interactions regulate immune cell recruitment, cytokine balance, endothelial permeability, and ECM deposition, while, in turn, immune and endothelial cells modulate mesothelial activation, proliferation, and transition. Understanding this complex network of intercellular communication provides new insights into fibrosis pathogenesis and reveals promising targets for antifibrotic therapies.

Vascular dysfunction lies at the core of cardiovascular diseases—the leading cause of global morbidity and mortality. Despite their prevalence, therapeutic options remain limited, in part due to an incomplete understanding of the molecular mechanisms driving vascular pathology. The integrated stress response (ISR), an evolutionarily conserved signaling network activated by diverse stressors, represents a critical but underexplored mechanism in vascular biology. This review examines the dual roles of the core ISR kinases—PERK, GCN2, HRI and PKR—in vascular homeostasis and pathology, including atherosclerosis, pulmonary hypertension, and angiogenesis. We develop a conceptual framework in which the ISR functions as a context-dependent, double-edged sword: while PERK and PKR promote inflammation, apoptosis, and vascular re-modeling, GCN2 mediates protective effects. The outcome of ISR activation is shaped by cell type, stress duration and intensity, and downstream signaling bias (e.g., ATF4 vs. CHOP dominance). We further discuss pharmacological ISR modulators—including 2-aminopurine, C16, salubrinal, halofuginone, GSK2606414, and GSK2656157—which have demonstrated beneficial effects in preclinical models by suppressing inflammation, reducing apoptosis, and attenuating disease progression. Collectively, the ISR emerges as a critical regulatory node in vascular pathophysiology, and its selective, context-aware modulation represents a promising avenue for therapeutic intervention.

This preprint has been withdrawn at the authors' request due to incomplete ethics statements concerning the use of primary human-derived cells.

Cardiospheres (CSs) are widely used to boost the pro-reparative potential of adult cardiac cells, mediated through their unique secretome profile. The original CS generation method relies on self-assembly of cardiac explant-derived cells (EDCs) on poly-D-lysine (PDL)-coated plates, but yields inconsistently sized spheroids, restricting broader applications. To address this, we employed ultra-low attachment (ULA) U-well plates to promote uniform spheroid assembly. We systematically compared CSs generated from mouse EDCs using the standard method, based on PDL-coated plates, and the alternative approach, based on ULA U-well plates. Both methods produced viable CSs mimicking the cardiac microenvironment, including mesenchymal cells/fibroblasts, smooth muscle, endothelial, and progenitor cells. PDL-formed CSs were characterized by size heterogeneity, increased stiffness, and reduced endothelial cell content. Despite that, they demonstrated elevated secretion of angiogenesis-related factors and robust proangiogenic potential in vivo. In contrast, generation of mCSs on ULA U-well plates resulted in the formation of soft spheroids with uniform size, enhanced vascularization (CD31+ cells), and increased MCP-1 secretion. In summary, the alternative U-well-based approach enables the generation of uniform spheroids with high spontaneous vascularization, while traditionally formed CSs using PDL-coated plates maintain their superior proangiogenic potential.

Vascular dysfunction lies at the core of cardiovascular diseases—the leading cause of global morbidity and mortality. Despite their prevalence, therapeutic options remain limited, in part due to an incomplete understanding of the molecular mechanisms driving vascular pathology. The Integrated Stress Response (ISR), an evolutionarily conserved signaling network activated by di-verse stressors, represents a critical but underexplored mechanism in vascular biology. This re-view examines the dual roles of the core ISR kinases—PERK, GCN2, HRI and PKR—in vascular homeostasis and pathology, including atherosclerosis, pulmonary hypertension, and angiogenesis. We advance a conceptual framework in which the ISR functions as a context-dependent, double-edged sword: while PERK and PKR promote inflammation, apoptosis, and vascular re-modeling, GCN2 mediates protective effects. The outcome of ISR activation is shaped by cell type, stress duration and intensity, and downstream signaling bias (e.g., ATF4 vs. CHOP dominance). We further discuss pharmacological ISR modulators—including 2-aminopurine, C16, salubrinal, halofuginone, GSK2606414, and GSK2656157—which have demonstrated beneficial effects in preclinical models by suppressing inflammation, reducing apoptosis, and attenuating disease progression. Collectively, the ISR emerges as a critical regulatory node in vascular pathophysiology, and its selective, context-aware modulation represents a promising avenue for therapeutic intervention.

Mesothelial cells (MCs) are highly relevant for studying the pathogenesis of serosal diseases, fibrosis, inflammation, and tumor progression. However, the isolation and maintenance of an epithelial-like phenotype of MCs in vitro remain methodologically challenging due to their tendency to undergo mesothelial-to-mesenchymal transition (MMT). In this work, we propose a combined protocol utilizing collagen IV coating, conditioned medium, and short-term ROCK inhibitor treatment, which improves cell survival. This approach enables the establishment of primary human cultures suitable for investigating mesothelial cell functional activity and for assessing the efficacy of potential therapeutic strategies.

Modern therapies aimed at stimulating heart vascularization are critical for regenerating damaged heart tissue and treating ischemic heart disease. Approaches based on developmental biology concepts, particularly those involving the use of cells to coordinate vascular network formation, are of great interest. In this context, epicardial mesothelial cells (MCs) have emerged as a key regulator of blood and lymphatic vessel development during cardiogenesis. However, therapeutic targeting of MCs remains challenging because of anatomical constraints and the difficulties related to isolation of viable cell cultures for research. In this study, we demonstrate for the first time that the pericardial fluid contains cell layers, being an easily accessible source of cardiac MCs. These cells exhibit a characteristic epithelial-like morphology and robust in vitro proliferation, and an ability to undergo epicardial-to-mesenchymal transition in response to TGFβ1. They secrete a broad range of proangiogenic and proinflammatory factors and exert a potent effect on endothelial cells, stimulating proangiogenic behavior and promoting vascular structure formation on Matrigel^TM. Treating MCs with TGF-β1 enhances the secretion of VEGF, G-CSF, GM-CSF and MCP-3, thereby boosting their proangiogenic properties. Therefore, pericardial fluid is an easily accessible source of MCs for studying their regulatory mechanisms, for being applied in tissue engineering, and for developing approaches to improve heart vascularization.