scRNA-seq reveals differential EC gene expression in endothelialized microfluidic devices exposed to a heterogeneous RBC suspension with a subpopulation of less-deformable RBCs compared with a homogeneous RBC suspension of hRBCs. Using the same 50 × 100 μm microfluidic device endothelialized to confluence with human umbilical vein ECs and RBC preparations as previously described (5% nystatin-treated with 95% hRBCs compared with 100% hRBCs), RBC suspensions were again perfused for 4 hours, but after the experiment, ECs were collected and prepared for scRNA-seq. (A) Uniform manifold approximation and projection (UMAP) embedding of scRNA-seq samples consisting of high-quality ECs portioned into 8 clusters with unique transcriptomic profiles. (B) Distribution of EC clusters after healthy vs nystatin-treated, low deformability RBC exposure. After exposure to nystatin-treated RBCs, a reduction in clusters C and D with a concomitant increase in clusters E and F was observed. (C) A heat map displaying the top markers expressed within each cluster. Columns represent individual cells, grouped by clusters, and rows display individual genes with the highest differential expression within each subcluster. Color bar shows gene expression levels, with red being high and blue being low gene expression. hRBC-enriched clusters C and D display an upregulation of genes associated with cellular proliferation and migration, including FP671120 (miR-3648), COL4A1 (collagen type IV), MKI67 (Ki-67), and CKS1B (CDC28 protein kinase regulatory subunit 1B). In contrast, clusters E and F, associated with nystatin-treated RBC exposure, demonstrate an upregulation of stress response genes, such as SRXN1 (sulfiredoxin 1) and ERG1 (early growth response protein 1), as well as several key regulatory factors involved in endothelial activation, including ANKRD1 (cardiac ankyrin repeat protein), KLF4 (Krüppel-like factor 4), FOS, and JUN. (D) Pathways enriched in each EC cluster. Clusters C and D, which were reduced in ECs exposed to nystatin-treated RBCs, are notably enriched in cellular proliferation and motility pathways, namely those involving RHO GTPase, laminin, integrin, and ECM proteoglycans, in addition to cell cycle pathways. Clusters E and F, enriched following nystatin-treated RBC exposure, exhibited significant enrichment of cellular response and oxidative stress pathways, including iron uptake, NFE2L2 signaling, and ROS and RNS production, as well as enriched cellular response and regulatory activity via kinase and transcription factor activation pathways. (E) A volcano plot illustrating differential gene expression in ECs exposed to nystatin-treated, low-deformability RBCs vs hRBCs, in which genes upregulated after nystatin-treated RBC exposure show a positive fold change, whereas those upregulated after hRBC exposure show a negative fold change (P < .00001). Several notable genes were observed to be significantly upregulated after nystatin-treated RBC exposure such as known markers of endothelial activation after injury, including THBS1, MMP1, CXCL8, EEF1A1, KLF6, and FN1. MARCKSL1, a key regulator of endothelial remodeling in response to disturbed flows, was also found to be upregulated. Markers of ferroptosis, GPX4, ATF3, PTGS2, and FTH1 were also upregulated. ECM, extracellular matrix; GTPase, guanosine triphosphate

scRNA-seq reveals differential EC gene expression in endothelialized microfluidic devices exposed to a heterogeneous RBC suspension with a subpopulation of less-deformable RBCs compared with a homogeneous RBC suspension of hRBCs. Using the same 50 × 100 μm microfluidic device endothelialized to confluence with human umbilical vein ECs and RBC preparations as previously described (5% nystatin-treated with 95% hRBCs compared with 100% hRBCs), RBC suspensions were again perfused for 4 hours, but after the experiment, ECs were collected and prepared for scRNA-seq. (A) Uniform manifold approximation and projection (UMAP) embedding of scRNA-seq samples consisting of high-quality ECs portioned into 8 clusters with unique transcriptomic profiles. (B) Distribution of EC clusters after healthy vs nystatin-treated, low deformability RBC exposure. After exposure to nystatin-treated RBCs, a reduction in clusters C and D with a concomitant increase in clusters E and F was observed. (C) A heat map displaying the top markers expressed within each cluster. Columns represent individual cells, grouped by clusters, and rows display individual genes with the highest differential expression within each subcluster. Color bar shows gene expression levels, with red being high and blue being low gene expression. hRBC-enriched clusters C and D display an upregulation of genes associated with cellular proliferation and migration, including FP671120 (miR-3648), COL4A1 (collagen type IV), MKI67 (Ki-67), and CKS1B (CDC28 protein kinase regulatory subunit 1B). In contrast, clusters E and F, associated with nystatin-treated RBC exposure, demonstrate an upregulation of stress response genes, such as SRXN1 (sulfiredoxin 1) and ERG1 (early growth response protein 1), as well as several key regulatory factors involved in endothelial activation, including ANKRD1 (cardiac ankyrin repeat protein), KLF4 (Krüppel-like factor 4), FOS, and JUN. (D) Pathways enriched in each EC cluster. Clusters C and D, which were reduced in ECs exposed to nystatin-treated RBCs, are notably enriched in cellular proliferation and motility pathways, namely those involving RHO GTPase, laminin, integrin, and ECM proteoglycans, in addition to cell cycle pathways. Clusters E and F, enriched following nystatin-treated RBC exposure, exhibited significant enrichment of cellular response and oxidative stress pathways, including iron uptake, NFE2L2 signaling, and ROS and RNS production, as well as enriched cellular response and regulatory activity via kinase and transcription factor activation pathways. (E) A volcano plot illustrating differential gene expression in ECs exposed to nystatin-treated, low-deformability RBCs vs hRBCs, in which genes upregulated after nystatin-treated RBC exposure show a positive fold change, whereas those upregulated after hRBC exposure show a negative fold change (P < .00001). Several notable genes were observed to be significantly upregulated after nystatin-treated RBC exposure such as known markers of endothelial activation after injury, including THBS1, MMP1, CXCL8, EEF1A1, KLF6, and FN1. MARCKSL1, a key regulator of endothelial remodeling in response to disturbed flows, was also found to be upregulated. Markers of ferroptosis, GPX4, ATF3, PTGS2, and FTH1 were also upregulated. ECM, extracellular matrix; GTPase, guanosine triphosphate

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