Journal: Journal of Biomedical Optics

Article Title: Functionalized erythrocyte-derived optical nanoparticles to target ephrin-B2 ligands

doi: 10.1117/1.JBO.24.8.085002

Figure Lengend Snippet: Ephrin-B2 Western blot expression. Western blot showing the expression of ephrin-B2 in hDMVECs infected with an ephrin-B2 lentivirus compared to the ephrin-B2 expression level of hDMVECs not infected with the ephrin-B2 lentivirus. Beta-actin levels are shown as the internal loading control.

Article Snippet: Human dermal microvascular endothelial cells (hDMVECs) were cultured in endothelial cell basal medium (ECBM) with growth supplement (Cell Applications, Inc.) as previously described.

Techniques: Western Blot, Expressing, Infection, Control

Journal: Journal of Biomedical Optics

Article Title: Functionalized erythrocyte-derived optical nanoparticles to target ephrin-B2 ligands

doi: 10.1117/1.JBO.24.8.085002

Figure Lengend Snippet: Cellular fluorescence of control and ephrin-B2 hDMVECs incubated with F-NETs at various ρ * values. (a) Fluorescent images of control and ephbin-B2 hDMVECs after 40 min of incubation at 4°C with F-NETs solutions. All images are falsely colored with the blue and red corresponding to DAPI and ICG NIR emission from the NETs, respectively. Scale bars are 30 μ m . (b) Averaged fluorescence intensity ( I ¯ ) [see Eq. (3)] as a function of ρ * . Cells from 3 to 4 images were analyzed, resulting in 16 to 41 measurements for each combination of nanoparticles and cells. Statistical significance of p < 0.001 is denoted by ***. Only statistically significant populations with the same ρ * value of F-NETs are indicated. (c) Sigmoidal fit to the I ¯ values of the ephrin-B2 hDMVECs versus the ρ * value of F-NETs. Error bars in (b) and (c) represent SDs.

Article Snippet: Human dermal microvascular endothelial cells (hDMVECs) were cultured in endothelial cell basal medium (ECBM) with growth supplement (Cell Applications, Inc.) as previously described.

Techniques: Fluorescence, Control, Incubation

Journal: bioRxiv

Article Title: Single Cell Transcriptomics of Fibrotic Lungs Unveils Aging-associated Alterations in Endothelial and Epithelial Cell Regeneration

doi: 10.1101/2023.01.17.523179

Figure Lengend Snippet: (A) Volcano plots showing the distribution of differentially expressed genes in gCap EC clusters 1 and 2 compared to other gCap EC clusters. (B) Violin plots showing the expression genes enriched in cluster 1 and 2. (C) Heatmap showing average expression of glycolysis genes in quiescent (Q) versus activated (A) gCap EC clusters. (D, E) Ingenuity pathway analysis shows canonical pathways and upstream regulators enriched in clusters 1 and 2 relative to other gCap EC clusters. P values were generated in IPA using Fisher’s test (log2 FC ≤-0.1 or ≥0.1, p value ≤0.05). P value and activation z-score were used for plotting canonical pathways and activated upstream regulators respectively. (F, G) qPCR analyses of human lung microvascular ECs (HLMECs) treated with the LATS1/2 inhibitor TRULI and the siRNAs targeting YAP and TAZ for 48 hours. YAP activation in these cells partially recapitulates the gene expression signature observed in activated gCap ECs. Values are summarized as mean and SD and analyzed using a two-tailed Student’s t -test. (N = 3). (H) Immunofluorescence images showing genetically labeled gCap ECs (membrane-GFP) in uninjured lungs of Aplnr-CreER(T)-mTmG reporter mouse. Lungs from these mice were harvested ten days following the last dose of tamoxifen (five total doses). An antibody against the pan-endothelial cell marker PECAM-1 was used to visualize all lung ECs. PECAM-1 positive ECs from large vessels showed no GFP expression. (I) Schematic representation of the approach used to detect TrkB-expressing gCap ECs following bleomycin injury. gCap ECs (mGFP) were lineage labeled in Aplnr-CreER(T)-mTmG mice 15 days prior to bleomycin administration (Day 0). Sham and bleomycin-injured lungs were harvested 28 days post bleomycin delivery and subjected to immunofluorescence analysis. An antibody against TrkB was used to detect injured gCap ECs (Red). gCap ECs co-expressing GFP and TrkB (yellow) only emerged in injured lungs (n=2). (J). Immunofluorescence images showing TrkB-expressing cells in the lungs of young and aged mice 37 days post bleomycin challenge (n=2). (K) Schematic showing putative mechanisms implicated in of gCap EC remodeling in response to lung injury and during lung fibrosis resolution.

Article Snippet: Human lung microvascular endothelial cells (HLMECs) were purchased from Cell Applications (San Diego, CA, USA) or ANGIO-PROTEOMIE (Worcester, MA, USA) and maintained in endothelial cell growth basal medium supplemented with microvascular endothelial cell growth kit.

Techniques: Expressing, Generated, Activation Assay, Gene Expression, Two Tailed Test, Immunofluorescence, Labeling, Membrane, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Top differentially expressed genes in flow conditioned primary human lung microvascular endothelial cells (HLMVEC) following exposure to heparinase III (HepIII) relative to vehicle control. Messenger RNA was collected from confluent monolayers of HLMVEC that were conditioned with 15 dyn/cm 2 for 48 h followed by exposure to heparinase III 500 mU/mL or vehicle for 6 h while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Heatmap representing top 40 differentially expressed genes in HLMVEC between heparinase III and vehicle. Each treatment group contains n = 2 RNA samples that were combined from HLMVEC within two ibidi channel slides, thus representing a total of n = 4 per condition. Colors represent gene expression z-score with red corresponding to upregulated and blue to downregulated. (B) Volcano plot depicting differential gene expression between HLMVEC exposed to heparinase III (positive log2 fold change) and vehicle (negative log2 fold change). Red genes meet figure thresholds of p ≤ 1 × 10 −3 and log2 fold change ≥|1| for the purposes of visualization. (C) Expression of the flow-responsive genes Krüppel-like factor 2 and 4 ( KLF2 , 4 ), endothelial nitric oxide synthase ( NOS3 ) and solute carrier family nine isoform A3 regulatory factor 2 ( SLC9A3R2 ) is reduced following heparinase III treatment. (D) Expression of angiopoietin-2 ( ANGPT2 ), endothelial cell-specific molecule-1 ( ESM1 , also known as endocan), and thrombospondin ( THBS1 ), markers of endothelial cell activation, is increased following heparinase III treatment.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Control, Shear, Gene Expression, Expressing, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Targeted representation of gene set enrichment analysis (GSEA) in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells (HLMVEC) after 6-h exposure to vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). GSEA was performed using (A) GO: Biological Process and (B) KEGG datasets. Figure displays up to twenty pathways from GSEA that are most relevant to endothelial cell organization and function with lowest False discovery rate (FDR)-adjusted p values (FDR q value). Pathways were organized according to their contribution to cellular maintenance and bioenergetics; cell organization and adhesion; angiogenesis and wound healing; or response to biophysical cues. Pathways on presented on the left were enriched in HLMVEC after exposure to vehicle whereas pathways on the right were enriched in HLMVEC after exposure to heparinase III. Circle size corresponds with number of genes present in experimental samples that overlap with respective dataset pathways, and circle shading represents the −log10 (FDR q value) with darker shades representing lower q values.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Differentially expressed genes that govern synthesis of heparan sulfate proteoglycans and glycosaminoglycans in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells treated for 6 h with vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Of the heparan sulfate proteoglycans found in the vascular endothelial apical glycocalyx, expression of syndecan 3 ( SDC3 ) and SDC4 were downregulated by heparinase III treatment. (B) Of the enzymes regulating hyaluronan expression in the endothelial glycocalyx, hyaluronan synthase isoform 2 ( HAS2 ) was upregulated while hyaluronidases 1 and 2 ( HYAL1 , 2 ) were downregulated by heparinase III treatment. (C) Of the enzymes that synthesize chondroitin sulfate expressed in the endothelial glycocalyx (commonly observed in SDC1 and SDC3) and that modify its sulfation, chondroitin sulfate synthase isoform 3 ( CHSY3 ) and chondroitin sulfate N -acetylgalactosaminylsulfotransferase isoform 1 ( CSGALNACT1 ) were upregulated while carbohydrate sulfotransferase isoform 15 ( CHST15 , catalyzing 6- O -sulfation of 4- O -sulfated N -acetylgalactosamine in chondroitin sulfate disaccharides) was downregulated following heparinase III treatment. (D) Of the enzymes that synthesize and modify heparan sulfate expressed in the endothelial glycocalyx, expression of N -deacetylase/ N -sulfotransferase isoform 1 ( NDST1 ) and glucuronic acid C5-epimerase ( GLCE ) (which also contributes to glucuronic acid epimerization to iduronic acid in chondroitin sulfate) were downregulated while heparan sulfate 3- O -sulfotransferase isoform 1 ( HS3ST1 ) and heparan sulfate 6- O -sulfotransferase isoform 3 ( HS6ST3 ) were upregulated following heparinase III treatment. We also found that heparinase III treatment suppressed heparanase ( HPSE ) expression. False discovery rate (FDR)-adjusted p values (FDR q values) are presented.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear, Expressing, Histone Deacetylase Assay