Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation

doi: 10.3389/fbioe.2022.903907

Figure Lengend Snippet: Effects of the presence (BM) versus the absence (NBM) of basement membrane proteins on human aortic endothelial cell (hAECs) migration and proliferation. Inverted microscopy images of GFP-labeled hAECs demonstrate that cells seeded on the BM surface cover a larger surface area than those seeded on the non-basement membrane protein (NBM) surface following 20 days in culture (A) . Dashed lines for Day 1 images indicate the initial seeding location of the cells. Dashed lines on Day 20 images indicate the final location of the cells. Due to the uneven surface of the scaffolds and the imaging method (stitch function in microscope Nikon Eclipse Ts2R), different values of cell brightness and focus are present in the images. A quantitative analysis of cell migration distance shows a significant increase in the maximal migration distance for cells seeded on the BM surface versus the NBM surface at all post-seeding timepoints (B) . Analysis of cell migration only (i.e., proliferation inhibited with mitomycin) shows a significant difference in the maximal radial distance migrated by treated-hAECs seeded on the BM surface (BM mitomyocin) when compared to treated-hAECs seeded on the NBM surface (NBM mitomyocin) at all timepoints (C) . Groups not connected by the same lower case letter are significantly different. Maximal migratory distance following 9 days of culture was reduced by the addition of mitomycin for cells seeded on the BM surface, but not for those seeded on the NBM surface (D) Assessment of proliferation only (i.e., FUCCI staining) demonstrated a significantly higher number of proliferating hAECs on the BM surface when compared to the cells in the NBM surface (D) . Scale bar A, 5,000 µm * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Human aortic endothelial cells (hAECs), GFP-labeled hAECs (GFP-hAECs), human umbilical vein cells (HUVECs), and GFP-labeled HUVECS (GFP-HUVECs, all cells from Angio-Proteomie, Boston, MA) were expanded to P4, unless otherwise stated, in a T-25 flask coated with quick coating solution (Angio-Proteomie) at 37 ° C, 5% CO 2 .

Techniques: Membrane, Migration, Inverted Microscopy, Labeling, Imaging, Microscopy, Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation

doi: 10.3389/fbioe.2022.903907

Figure Lengend Snippet: Effects of the presence (BM) versus the absence (NBM) of basement membrane proteins on hAEC phenotypes and functions. Representative images of immunofluorescent staining of human laminin production demonstrates the increased production of human laminin in the hAECs seeded on the non-basement membrane protein (NBM) surface when compared to cells seeded on the BM surface (A) . A quantitative analysis of the human laminin production per cell shows a significant higher production of human laminin by the hAECs seeded on the NBM surface versus on the BM surface (D) . Representative images of immunofluorescent staining for endothelial cell polarization demonstrates that the BM presence mediates the polarization of podocalyxin (green) to the apical cell surface of seeded hAECs, indicating their quiescent phenotype (B) . Conversely, apical polarization of podocalyxin is not present on hAECs seeded on the NBM protein side (B) . Similarly, representative images of the adherence junction immunofluorescent staining for VE-cadherin (red) and β-catenin (green) resulted in appropriate expression and co-localization of these adherence junction proteins (indicated by the arrow) in hAECs seeded on the BM surface, while expression and co-localization is not seen in cells seeded on the NBM protein surface (C) . A quantitative analysis of nitrate and nitrite secretions (nitric oxide biproducts) of HUVEC-seeded AR-scaffolds, show a significant increase in nitrate and nitrite secretions by cells seeded on the BM protein surface, compared to the cells seeded on the NBM protein surface or tissue culture plastic (E) . Scale bar A, 100 µm. Scale bars B and C, 10 µm * = p < 0.05, ** = p < 0.01.

Article Snippet: Human aortic endothelial cells (hAECs), GFP-labeled hAECs (GFP-hAECs), human umbilical vein cells (HUVECs), and GFP-labeled HUVECS (GFP-HUVECs, all cells from Angio-Proteomie, Boston, MA) were expanded to P4, unless otherwise stated, in a T-25 flask coated with quick coating solution (Angio-Proteomie) at 37 ° C, 5% CO 2 .

Techniques: Membrane, Staining, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation

doi: 10.3389/fbioe.2022.903907

Figure Lengend Snippet: Differential gene expression and the gene set enrichment analysis of scaffold seeded, simvastatin-, and TNFα-treated hAECs. (A) Heatmap of all 415 identified significantly differentially expressed genes across all three treatment groups. (B–D) Significantly enriched pathways in the KEGG and HALLMARK gene sets following GSEA for simvastatin (B) , scaffold (C) , and TNFα (D) groups. (E–G) Representative GSEA plots across all three groups for three biologically relevant pathways, including mitosis (E) , programmed cell death (F) , and glucose metabolism (G) .

Article Snippet: Human aortic endothelial cells (hAECs), GFP-labeled hAECs (GFP-hAECs), human umbilical vein cells (HUVECs), and GFP-labeled HUVECS (GFP-HUVECs, all cells from Angio-Proteomie, Boston, MA) were expanded to P4, unless otherwise stated, in a T-25 flask coated with quick coating solution (Angio-Proteomie) at 37 ° C, 5% CO 2 .

Techniques: Gene Expression

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation

doi: 10.3389/fbioe.2022.903907

Figure Lengend Snippet: tSNE clustering analysis of scaffold seeded, simvastatin, and TNFα-treated hAECs. The tSNE clustering analysis plot is driven by the top 1,000 variable genes across all samples (A) . PC1 accounts for 79.88% of the variance, while PC2 explains the remaining 20.22%. Heatmap of the top 100 genes responsible for driving PC1 (B) . PC1 is almost exclusively driven by up-regulated genes in the TNFα group. Enrichment plot of the top 10 enriched gene ontology biological processes for the top 100 genes driving PC1 (D) . The top 10 enriched pathways in PC1 are all associated with pro-inflammatory responses. Heatmap of the top 100 genes responsible for driving PC2 (C) . PC2 is predominantly driven by up-regulated genes in the scaffold group. Enrichment plot of the top 10 enriched gene ontology biological processes for the top 100 genes driving PC2 (E) . 50% of the enriched pathways in PC2 are associated with cellular chemotaxis and respond to chemokines.

Article Snippet: Human aortic endothelial cells (hAECs), GFP-labeled hAECs (GFP-hAECs), human umbilical vein cells (HUVECs), and GFP-labeled HUVECS (GFP-HUVECs, all cells from Angio-Proteomie, Boston, MA) were expanded to P4, unless otherwise stated, in a T-25 flask coated with quick coating solution (Angio-Proteomie) at 37 ° C, 5% CO 2 .

Techniques: Chemotaxis Assay

Journal: Scientific Reports

Article Title: Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

doi: 10.1038/s41598-019-53230-1

Figure Lengend Snippet: ECM niche modulates differential morphology of seeded hAEC. ( A – D ) Representative images of eGFP-hAEC seeded on fibrous or serous side of 6 mm AR-BP discs and imaged after 2 ( A , C ) and 4 ( B , D ) days show that higher number of cells can be observed on serous side compare to fibrous. ( E – H ) When seeded at higher densities and cultured for 2 days, eGFP-hAEC (( E ) 71 cells/mm 2 ; ( F ) 142 cells/mm 2 ; ( G ) 284 cells/mm 2 , ( H ) 568 cells/mm 2 ) seeded on the serous side show multiple vacuoles (arrows) and rounded morphology compared to fibrous side. ( I ) Quantification of cell aspect ratio demonstrates that cells seeded on serous side are statistically more rounded (lower aspect ratio) than those seeded on the fibrous side, and that cell morphology becomes increasingly more rounded with higher seeding densities regardless of seeding side. Scale bar 1 mm ( A , B ), 200 μm ( C – H ). Data represent the mean values natural log-transformed aspect ratios ± SD, n = 5 per group per seeding density (****p < 0.0001).

Article Snippet: Enhanced green fluorescent protein (eGFP) transfected hAEC were purchased from Angio-Proteomie (Boston, MA) (CAT No. cAP‐0006GFP, LOT No. 201209701SR).

Techniques: Cell Culture, Transformation Assay

Journal: Scientific Reports

Article Title: Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

doi: 10.1038/s41598-019-53230-1

Figure Lengend Snippet: Actin expression in eGFP-hAEC seeded on AR-BP scaffolds. Representative microscopic images (n = 5 per group) of eGFP-hAEC (green, first column) after 4 days proliferation and staining for Actin (Phaliodin-594; red, second column) and nuclei (Hoechst 33342; blue, third column). ( A , B ) 71 cell/mm 2 ; ( C , D ) 142 cell/mm 2 ; ( E , F ) 284 cell/mm 2 and ( G , H ) 568 cell/mm 2 . Scale bars: 100 μm (first to fourth column) and 20 μm (fifth column). ( I ) Quantification of cell number on the serous and the fibrous side 4 days after seeding at different densities demonstrate higher cell numbers in the serous side for low and moderate seeding densities (71, 142, and 284 cells/mm 2 ), while cell number equalize when seeded at very high densities (568 cells/mm 2 ). Data represent the average number of cells in a 1.6 mm 2 area ± SD, n = 5 images per group (seeding density and side) (**p < 0.01, *p < 0.05, n.s. non-significant p = 0.4).

Article Snippet: Enhanced green fluorescent protein (eGFP) transfected hAEC were purchased from Angio-Proteomie (Boston, MA) (CAT No. cAP‐0006GFP, LOT No. 201209701SR).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

doi: 10.1038/s41598-019-53230-1

Figure Lengend Snippet: ECM niche modulates hAEC human laminin production following seeding on AR-BP scaffolds. Representative fluorescent microscopy images of ( A’ – D’ ) cell nuclei (blue), ( A” – D” ) eGFP-hAEC (green), and ( A”’ – D”’ ) human laminin (red). The serous side was found to have a continuous laminin layer while on the fibrous side new laminin production was found to be strongly associated with areas of cellular adhesion, especially at low seeding densities (n = 5 per group). ( E , F ) Quantification of human laminin production per cell. At moderate seeding density (142 cells/mm 2 ) hAEC seeded on the serous side produce more laminin per cell than those seeded on the fibrous side (p = 0.0002). At high seeding density (568 cells/mm 2 ) the effect of ECM niche on per cell laminin production is no longer present. Scale bar = 10 µm.

Article Snippet: Enhanced green fluorescent protein (eGFP) transfected hAEC were purchased from Angio-Proteomie (Boston, MA) (CAT No. cAP‐0006GFP, LOT No. 201209701SR).

Techniques: Microscopy