Itoh, F. | Search bioRxiv

bioRxiv2026

Three-dimensional vascular microenvironments uncover endothelial plasticity during TGF-β2-driven vascular remodeling

The tumor microenvironment plays a pivotal role in tumor development, harboring elements such as endothelial cells, immune cells, fibroblasts, and soluble factors such as transforming growth factor-{beta} (TGF-{beta}) family. TGF-{beta} family regulates cell development and promotes tumor invasion, metastasis, angiogenesis, and endothelial-to-mesenchymal transition (EndoMT). Here, we investigate the effects of TGF-{beta} signaling on vascular remodeling using a three-dimensional (3D) vascular network in a microfluidic device. Using both a co-culture (3D-Co) and simplified endothelial monoculture (3D-CM), we demonstrate that TGF-{beta} signaling reduces the quality and functionality of the vasculature by regressing them. In addition, we observed the upregulation of EndoMT-related markers in mRNA and protein expressions, suggesting the induction of EndoMT in 3D vascular networks. The increased vascular permeability stimulated by TGF-{beta}2 also supports the loss of endothelial identity in the 3D-Co. Transcriptomic analysis revealed the coordinated activation of pathways associated with cell migration and EndoMT, along with the suppression of cell cycle progression. A comparative analysis of two-dimensional (2D) and 3D cultures revealed a fundamentally distinct endothelial response to TGF-{beta}2 in the 3D context, including metabolic reprogramming. These findings demonstrate that the 3D microenvironment critically modulates endothelial responses to TGF-{beta} and enables the emergence of vascular phenotypes not captured in 2D systems. This study provides a more physiologically relevant platform to investigate endothelial dysfunction and vascular remodeling.

bioengineering↗

bioRxiv2024

Atheroprone Flow Activates SMAD-FOXO1 to drive Endothelial-to-Mesenchymal Transition and Atherosclerosis

BackgroundCardiovascular diseases are the leading cause of death worldwide with atherosclerosis as the main underlying pathology. A hallmark of atherosclerotic lesion formation is endothelial-to-mesenchymal transition (EndoMT) triggered by perturbed blood flow patterns at arterial bifurcations and curvatures. SMAD transcription factors (TFs), activated by bone morphogenetic protein (BMP) 9/10 or transforming growth factor beta (TGF{beta}) signaling, are indispensable for endothelial homeostasis. Yet, they also play a significant role in stimulating EndoMT. How different interacting co-factors mediate the shift towards a pathological SMAD response remains elusive. MethodsWe generated endothelial cell (EC)-specific SMAD1/5 or SMAD2/3 knock-out mice and performed assay for transposase accessible chromatin sequencing (ATAC-Seq) of EC nuclei from regions of atheroprone (aortic arch) and atheroprotective (descending thoracic aorta) flow to identify transcriptional co-regulators of SMADs. We validated this using single-cell (sc)ATAC-Seq and immunofluorescence staining data from wild-type mice. To assess conservation of our findings for the human situation, we performed co-immunoprecipitation and proximity ligation assays in human aortic ECs (HAoECs). We exposed HAoECs to pathological or physiological (i.e. oscillatory or pulsatile) flow and performed ATAC- and RNA-Seq. Next, transcriptomic and chromatin accessibility data were integrated and motif enrichment and TF footprinting analysis were performed. Finally, we used siRNA-mediated approaches, TF inhibition, and luciferase-based reporter gene assays to analyze the transcriptional response of target TFs and explored their presence in plaques of atheroprone low-density lipoprotein receptor-deficient mice. ResultsWe observed enrichment of FOXO TF family motifs in DNA loci with increased accessibility in response to atheroprone flow in vitro and in vivo. These motifs were associated with genes displaying enhanced mRNA expression. We observed that FOXO motifs are enriched in peaks lost upon EC-specific SMAD KO in mice. We identified SMADs and FOXO1 as interacting partners that form complexes upon atheroprone flow stimulation. Inhibitor experiments revealed that FOXO1 and SMADs mediate EndoMT upon atheroprone flow exposure by upregulating SNAI2. ConclusionWe identified SMAD/FOXO1 complexes that mediate EndoMT in response to atheroprone flow. Targeting this interaction can potentially reduce atherosclerotic burden by interfering with pathological flow-induced EndoMT and thus disease progression.

genomics↗