Biology subjects

Yordanov, T. E.

Publications and source records attributed to Yordanov, T. E..

4 recordsLinked to original sources

bioRxiv2025

Animal-Origin-Free Method for Generating Blood Vessel Organoids

BackgroundBlood vessel organoids (BVOs) represent a promising tool for modeling vascular diseases, drug screening, and regenerative therapies. However, current protocols for BVO generation are complex, labor-intensive, and reliant on animal-derived extracellular matrices (ECM) such as Matrigel, limiting reproducibility, scalability, and clinical applicability. MethodsWe developed a simplified, animal-origin-free protocol for BVO generation that addresses current limitations and enables high-throughput automated workflows. The method employs ultra-low attachment 96-well U-bottom plates for standardized aggregation and differentiation of human induced pluripotent stem cells (hiPSCs) in a human derived collagen-based extracellular matrix. Unlike conventional protocols where aggregates are embedded in a two-layer ECM, our approach utilizes a single-layer, which we termed "sitting drop". This innovative approach requires considerably fewer materials and handling steps and is compatible with high-throughput automated machines. ResultsBVO generation utilizing the here described optimized protocol resulted in the formation of BVOs with reproducible morphology and cellular composition. Flow cytometry confirmed the presence of CD31 endothelial cells and PDGFR{beta} pericytes in BVOs, generated in sitting drops, with cell population percentages comparable to those observed in traditional two-layer BVO cultures. In vivo transplantation of mature BVOs in a mouse full-thickness skin wound model demonstrated successful integration of BVO derived cells into host vessels, highlighting their potential in cell-based therapies. ConclusionOur study presents a robust and animal-origin-free method for BVO generation based on single-layer "sitting drop" cultures. This protocol maintains cellular integrity while enhancing reproducibility and automation-readiness, paving the way for high-throughput screening and clinical translation of vascular organoid technology. HighlightsO_LIEntire blood vessel organoid (BVO) workflow performed in a single ultra-low attachment-96 plate C_LIO_LIFully animal-origin-free: no Matrigel or Geltrex required throughout the protocol C_LIO_LIRobust generation of BVOs using human collagen-based ECM C_LIO_LIHigh-throughput compatible and automation-ready "sitting drop" culture system C_LIO_LIIntegration of BVO-derived cells into host vessels in vivo C_LI

bioengineering↗

bioRxiv2025

Integrin β1-Talin1 at focal adhesions underpin uncontrolled endothelial cell enlargement in live cerebral cavernous malformation vasculature

Cerebral cavernous malformations (CCMs) are vascular anomalies caused by loss of CCM gene function and consequent hyperactivation of MEKK3-KLF2/4 signaling in endothelial cells. Excess integrin {beta}1 activity has been associated with lesion growth, yet the precise mechanistic and biomechanical roles of the integrin-KLF2/4 hierarchy in CCM cellular pathogenesis are not fully understood. Using live imaging of endothelial Vinculin in Ccm1-deficient zebrafish, we demonstrate excessive, mechanically active focal adhesions in an in vivo model of CCM pathology. We validate this in CCM1-deficient endothelial cells and show a redistribution of mechanical tension from cell-cell junctions to focal adhesions. Genetic deletion of Talin1 to decouple focal adhesions from the cell cortex and inhibit integrin {beta}1 signaling demonstrates the integrin {beta}1-Talin1 complex is essential for vascular malformations in ccm1 mutants by driving endothelial cell enlargement. We show integrin {beta}1-Talin1 act independent or downstream of KLF2/4, rather than upstream as previously suggested. Thus, we reposition the role of integrin {beta}1-Talin1 in CCM pathogenesis, demonstrating an essential function in driving cell enlargement, which underpins lesion growth.

cell biology↗

bioRxiv2023

Cerebral Cavernous Malformation severity is impacted by distinct forms of Hyaluronic acid in the vascular microenvironment

Cerebral Cavernous Malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system (CNS) upon loss of the CCM multimeric protein complex. The endothelial cells (ECs) within CCM lesions are characterised by overactive MEKK3 kinase and KLF2/4 transcription factor signalling, leading to pathological changes such as increased EC spreading and reduced junctional integrity. Concomitant to aberrant EC signalling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the CNS. Here, we adapted a three dimensional (3D) microfluidic system to examine CCM1 deficient human micro-vessels in distinctive ECMs. We validate that EC pathological hallmarks are maintained in this 3D model. We further show that key genes responsible for homeostasis of Hyaluronic Acid (HA), a major ECM component of the CNS, are dysregulated in CCM. Supplementing the ECM in our model with forms of HA that are predicted to be reduced, inhibits CCM cellular phenotypes, independent of KLF2/4. This study thereby provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signalling impact vascular malformations.

cell biology↗

bioRxiv2022

Dynamically regulated Focal adhesions coordinate endothelial cell remodelling in developing vasculature

The assembly of a mature vascular network involves the coordinated control of cell shape changes to regulate morphogenesis of a complex vascular network. Cellular changes include a process of endothelial cell (EC) elongation which is essential for establishing appropriately sized lumens during vessel maturation1-3. However how EC elongation is dynamically regulated in vivo is not fully understood since live monitoring of this event can be challenging in animal models. Here, we utilise the live imaging capacity of the zebrafish to explore how integrin adhesion complexes, known as Focal Adhesions (FAs), control EC dynamics in live flow pressured vasculature. To do this, we generated a zebrafish mutant, deficient for the integrin adaptor protein Talin1. Notably, unlike the severe cardiovascular defects that arise in Talin1 knockout mice4, vasculogenesis still occurs normally talin1 mutants and cardiac output remains sufficient up to two days post fertilisation (dpf). This allowed us to uncouple primary roles for FAs in ECs during subsequent morphogenesis events, including angiogenesis and vessel remodelling, without interference of secondary effects that might occur due to systemic vessel failure or loss of blood flow. We further established a FA marker line, expressing endothelial Vinculin-eGFP, and demonstrated that FAs are lost in our talin1 mutants. This Vinculin transgene represents the first in vivo model to monitor endothelial FA dynamics. Loss of FAs in talin1 mutants, leads to compromised F-actin rearrangements, which perturb EC elongation and cell-cell junction linearisation during vessel remodelling. Chemical induction of actin polymerisation can restore these cellular phenotypes, suggesting a recovery of actin rearrangements that are sufficient to allow cell and junction shape changes. Together, we have identified that FAs are essential for active guidance of EC elongation and junction linearisation in flow pressured vessels. These observations can explain the severely compromised vessel beds, haemorrhage and vascular leakage that has been observed in mouse models that lack integrin signalling4-8.

cell biology↗