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Introduction Endothelial cell injury and endothelial-to-mesenchymal transition (EndMT) are key mechanisms of vasculopathy in systemic sclerosis (SSc). Yet, development and testing of new therapies remain hindered by the scarcity of suitable mouse models and the lack of complex in vitro systems that accurately replicate SSc vasculopathy.In this study, we aimed to investigate whether blood vessel organoids (BVOs) generated from induced pluripotent stem cells (iPSCs) could serve as a novel three-dimensional multicellular model to explore SSc vasculopathy under defined experimental conditions.Material and Methods BVOs were generated from iPSCs of healthy individuals and SSc patients treated with serum from healthy donors or SSc patients. Parameters of angiogenesis were quantified using AngioTool. Proteomic changes, transcriptomic changes and chromatin accessibility were assessed by CODEX multiplex imaging, RNA-seq and ATAC-seq, respectively. Bosentan (BST) and the γ-secretase inhibitor DAPT were tested for their effects in preventing angiogenesis defects in BVOs.Results SSc BVOs treated with SSc serum demonstrated impaired angiogenesis compared to SSc BVOs treated with healthy serum and to healthy BVOs.RNA-seq, ATAC-seq and CODEX imaging revealed concerted epigenomic, transcriptomic and proteomic changes with upregulation of genes, proteins and pathways involved in extracellular matrix adhesion, collagen synthesis and EndMT, alongside downregulation of tight junction-associated pathways and genes and proteins related to an endothelial phenotype in SSc BVOs upon treatment with SSc serum.CODEX imaging demonstrated that BVOs recapitulate endothelial cell and pericyte populations and their changes in frequencies in SSc. Two subpopulations of endothelial cells showed high expression of the EndMT marker αSMA and were highly upregulated in SSc BVOs upon exposure to SSc serum, thus recapitulating the upregulation of the αSMA+ endothelial cells described previously by our group in SSc. These populations were spatially avoiding pericytes upon exposure to SSc serum, despite their increased frequency, which contributed to the angiogenic defects.Treatment with BST and DAPT prevented the angiogenesis defects induced by SSc serum in SSc BVOs.Conclusions We show that SSc BVOs recapitulate key features of SSc vasculopathy, including impaired angiogenesis and enhanced EndMT, increases in pathologic endothelial cell populations, as well as loss of endothelial cells-pericytes interactions when exposed to SSc serum. We further validate that BVOs can be used for drug testing in SSc, and employ them to demonstrate that inhibition of Notch signaling might be a novel approach for targeting vasculopathy in SSc.