Journal: PLoS ONE

Article Title: Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-β/ALK5 signaling

doi: 10.1371/journal.pone.0244243

Figure Lengend Snippet: ( A ) Representative images of microvascular tubules in 3:1 and 3:3 MVEC:AoAF co-cultures in degradable 7.5wt% hydrogels with 3 mM RGD after 7, 14, and 28 days of culture. MVECs are depicted by CD31 (magenta), while F-actin is stained with phalloidin-568 (yellow) and nuclei are counterstained with Hoechst 33258 (cyan). Scale bar = 50 μm. ( B-F ) Quantification of network parameters including: ( B ) number of tubules, ( C ) number of junctions, ( D ) total tubule length, ( E ) tubule diameter. ( F-G ) Representative images MVEC microvessels in ( F ) 3:1 and ( G ) 3:3 co-cultures in 7.5wt% hydrogels with 3 mM RGD after 14 days of culture. Z-stack cross-sections demonstrate the formation of a hollow lumen structure in 3:3 co-cultures. MVECs are depicted by CD31 (magenta) and nuclei are counterstained with Hoechst 33258 (cyan). Scale bar = 50 μm. In B-E : data are represented as the mean ± SEM, with n = 3 biological replicates per condition. A two-way ANOVA, followed by a Tukey HSD post hoc test, was used to detect statistical significance, *p<0.05 for a given time point relative to day 7, within an individual co-culture, # p<0.05 for 3:3 co-cultures compared to 3:1 co-cultures for a given time point.

Article Snippet: MVECs (35 yo male donor; Lonza, Walkersville, MD) were cultured in microvascular endothelial cell growth medium-2 (EGM2-MV), which comprised microvascular endothelial cell basal medium supplemented with 5% fetal bovine serum (FBS), and a proprietary cocktail of hydrocortisone, basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor, insulin-like growth factor 1, ascorbic acid, and epidermal growth factor (all from Lonza), and used between passage numbers 6 and 8 for all assays.

Techniques: Staining, Co-Culture Assay

Journal: PLoS ONE

Article Title: Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-β/ALK5 signaling

doi: 10.1371/journal.pone.0244243

Figure Lengend Snippet: ( A ) To determine whether growth factors secreted by AoAFs promoted MVEC viability and spreading in degradable PEG hydrogels, encapsulated MVECs were cultured for 7 days in either fresh co-culture medium or AoAF conditioned medium. MVEC viability was high (>80%) after 1 day of encapsulation; however, viability significantly decreased after 7 days of culture in either fresh co-culture medium or AoAF conditioned medium. ( B ) To investigate whether direct contact between AoAFs and MVECs was necessary for directing microvessel formation, MVECs and AoAFs in separate layers of a hydrogel. MVECs were encapsulated in a layer of enzymatically degradable hydrogel (7.5wt% hydrogel with 3 mM RGD) and AoAFs were encapsulated in a layer of non-degradable hydrogel (10wt% hydrogel with 3 mM RGD) to limit direct contact of the two cell types. ( C ) Viability of encapsulated cells in the layered hydrogel system was high (>80%) after 24 hrs. AoAFs remained highly viable on day 7, while MVEC viability was significantly reduced. ( D ) However, when MVECs and AoAFs were encapsulated in separate layers of enzymatically degradable hydrogel, ( E ) MVEC viability was stable after 7 days of culture and ( F ) MVEC tubules formed at the intersection of degradable hydrogel layers after 14 days of culture. MVECs are depicted by CD31 (magenta), while F-actin is stained with phalloidin-568 (yellow) and nuclei are counterstained with Hoechst 33258 (cyan). Scale bar = 50 μm. In A, C, & E : data are represented as the mean ± SEM, with n = 3 biological replicates per condition. A two-way ANOVA, followed by a Tukey HSD post hoc test, was used to detect statistical significance, *p<0.05 for day 7 relative to day 1, within an individual cell system; ns = no significance.

Article Snippet: MVECs (35 yo male donor; Lonza, Walkersville, MD) were cultured in microvascular endothelial cell growth medium-2 (EGM2-MV), which comprised microvascular endothelial cell basal medium supplemented with 5% fetal bovine serum (FBS), and a proprietary cocktail of hydrocortisone, basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor, insulin-like growth factor 1, ascorbic acid, and epidermal growth factor (all from Lonza), and used between passage numbers 6 and 8 for all assays.

Techniques: Cell Culture, Co-Culture Assay, Encapsulation, Staining

Journal: PLoS ONE

Article Title: Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-β/ALK5 signaling

doi: 10.1371/journal.pone.0244243

Figure Lengend Snippet: ( A ) Representative images of microvascular tubules in 3:3 MVEC:AoAF co-cultures in degradable 7.5wt% hydrogels with 3 mM RGD after 14 days of culture with 1 μM A83-01 (ALK5 inhibitor) or DMSO (control). MVECs are depicted by CD31 (magenta), while F-actin is stained with phalloidin-568 (yellow) and nuclei are counterstained with Hoechst 33258 (cyan). Scale bar = 100 μm. The addition of 1 μM A83-01 to 3:3 co-cultures resulted in significantly decreased ( B ) number of junctions per mm 2 , ( C ) number of MVEC tubules per mm 2 , ( D ) total tubule length (mm/mm 2 ), ( E ) tubule diameter, compared to control co-cultures on day 14. ( F ) Following treatment with 1 μM A83-01, the number of total cells, MVECs, and AoAFs per mm 3 was decreased compared to control cultures on day 14. Data are normalized to day 1 (dashed line). ( G ) Treatment with 1 μM A83-01 significantly reduced the production of the basal lamina proteins, collagen IV (green) and laminin (green), surrounding MVECs (magenta) compared to control co-cultures on day 14. Nuclei are counterstained with Hoechst 33258 (blue). Scale bar = 100 μm. Quantification of ( H ) the number AoAF interactions per mm 3 and ( I ) the number AoAF interactions per tubule in co-cultures treated with 1 μM A83-01 or DMSO controls. ( J ) A low percentage of αSMA + AoAFs were observed in 1 μM A83-01 co-cultures after 14 days and ( K ) were generally unaligned with MVEC tubules. ( L ) Representative images of αSMA + AoAFs (green) in -cultures treated with 1 μM A83-01 on day 14. F-actin is stained with phalloidin-568 (yellow), nuclei are counterstained with Hoechst 33258 (cyan), and MVECs are labeled with CD31 (magenta). Scale bar = 50 μm. In B-F, H-K : data are represented as the mean ± SEM, with n = 3 biological replicates per condition. In B-E, H-K : an unpaired student’s t-test detect statistical significance, *p<0.05 for hydrogels treated with 1 μM A83-01 relative to control hydrogels. In F : a two-way ANOVA, followed by a Tukey HSD post hoc test, was used to detect statistical significance, *p<0.05 for hydrogels on day 14 relative day 1, # p<0.05 for hydrogels treated with 1 μM A83-01 relative to control hydrogels on day 14.

Article Snippet: MVECs (35 yo male donor; Lonza, Walkersville, MD) were cultured in microvascular endothelial cell growth medium-2 (EGM2-MV), which comprised microvascular endothelial cell basal medium supplemented with 5% fetal bovine serum (FBS), and a proprietary cocktail of hydrocortisone, basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor, insulin-like growth factor 1, ascorbic acid, and epidermal growth factor (all from Lonza), and used between passage numbers 6 and 8 for all assays.

Techniques: Control, Staining, Labeling

Journal: Advanced healthcare materials

Article Title: Human Adventitial Fibroblast Phenotype Depends on the Progression of Changes in Substrate Stiffness

doi: 10.1002/adhm.201901593

Figure Lengend Snippet: (A) Cell area increased over time in 5wt% and 7.5wt% hydrogels, but not in 10wt% substrates. (B) Similarly, AoAF proliferation increased in 5wt% and 7.5wt% hydrogels over time. Data are mean DNA content relative to DNA content in hydrogels after 1 day of culture (dashed line) ± SEM, n = 3 biological replicates per condition. (C) The percentage of αSMA` AoAFs was significantly increased in 5wt% hydrogels over time, but alterations were not observed in 7.5wt% and 10wt% hydrogels. Expression of (D) collagen I and (E) collagen III, measured via corrected total cell fluorescence, by AoAFs encapsulated in 5wt%, 7.5wt%, and 10wt% hydrogels was altered over time. (F) Ratio of collagen I to collagen III, determined from total collagen signal obtained from the corrected total cell fluorescence, in AoAF-laden 5wt%, 7.5wt%, and 10wt% hydrogels over time. In A-F, data are represented as mean ± SEM, n = 3 biological replicates per condition. A two-way ANOVA, followed by a Tukey HSD post hoc test, was used to detect statistical significance, *p<0.05, **p<0.01.

Article Snippet: Human aortic adventitial fibroblasts (AoAF; deidentified 53 yo male donor; Lonza, Allendale, NJ) were cultured in stromal cell growth medium (SCGM), which comprised fibroblast basal medium supplemented with 5% FBS, basic fibroblast growth factor, and insulin (all from Lonza).

Techniques: Expressing, Fluorescence

Journal: Advanced healthcare materials

Article Title: Human Adventitial Fibroblast Phenotype Depends on the Progression of Changes in Substrate Stiffness

doi: 10.1002/adhm.201901593

Figure Lengend Snippet: (A) Alterations in swollen storage modulus, measured using oscillatory shear rheology, were observed in AoAF-laden 5wt%, 7.5wt%, and 10wt%, hydrogels over time. (B) Increased proteolytic activity was observed in hydrogels undergoing degradation (i.e. decreased bulk shear storage modulus. Data are mean proteolytic activity relative to acellular hydrogels (dashed line). (C) MMP-2 production by encapsulated AoAFs was similar among hydrogel formulations and over time. (D) Quantification of the percentage of MMP-9-positive cells in AoAF-laden 5wt%, 7.5wt%, and 10wt% hydrogels over the 42 day culture. (E-F) Quantification of the tissue inhibitors of metalloproteinases (E) TIMP-1 and (F) TIMP-2 in the medium collected from AoAF-laden 5wt%, 7.5wt%, and 10wt% hydrogels during the 42 day culture period. Data are mean ± SEM, where in A: n = 6 per condition, in B, D-F: n = 3 biological replicates per condition, and in C: n > 30 cells from 3 images from each of 3 hydrogels per condition. In A-F: a two-way ANOVA, followed by a Tukey HSD post hoc test, was used to detect statistical significance. In A-B: *p<0.01 for 10wt% relative to 5wt%, #p<0.05 for 10wt% relative to 7.5wt%, $p<0.05 for 7.5wt% relative to 5wt%, at a given time point; &p<0.01 for a given time point relative to day 1, within an individual hydrogel formulation. In C: no statistical significance (ns) was indicated following a two-way ANOVA. In D-F: *p<0.05 for indicated comparisons.

Article Snippet: Human aortic adventitial fibroblasts (AoAF; deidentified 53 yo male donor; Lonza, Allendale, NJ) were cultured in stromal cell growth medium (SCGM), which comprised fibroblast basal medium supplemented with 5% FBS, basic fibroblast growth factor, and insulin (all from Lonza).

Techniques: Shear, Activity Assay, Formulation