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

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: bioRxiv

Article Title: Extracellular Vesicle-Mediated Purinergic Signaling Contributes to Host Microenvironment Plasticity and Metastasis in Triple Negative Breast Cancer

doi: 10.1101/2020.09.11.293837

Figure Lengend Snippet: (A) Permeabilization of HLMVEC and (B) HUVEC monolayers to FITC-dextran was measured using Matrigel-coated tranwell chambers. Cells were treated with complete growth medium (VL-MV or VL-VEGF) with and without HME1 EVs, MDA-MB-231 EVs, and MDA-MB-231 EVs with MRS2179, EA, or a combination of both drugs. Intensity of FITC-dextran in top chambers was measured at indicated time points as an indicator of enhanced permeability. n = 6. Mean ± S.E.M. *** P < 0.001 by one-way ANOVA and Tukey post-test. (C) CLSM images of HLMVEC monolayers immuno-stained for actin, ZO-1 and β-catenin expression. All CLSM laser acquisitions were kept consistent, except for bottom two images on the right, where 488 nm laser was enhanced to better visualize cells in these two treatment conditions. Cells were counterstained with DAPI. Scale 100 μm.

Article Snippet: Primary human umbilical vein endothelial cell (HUVEC) and human lung microvascular endothelial cell (HLMVEC) lines (Lifeline Cell Technology, Frederick, MD) were grown in VascuLife-VEGF and Vasculife-MV media, respectively (Lifeline Cell Technology).

Techniques: Permeability, Staining, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: ER stress inhibition restores palmitate-dependent impairment of autophagy and induction of UPR in macro- and microvascular endothelial cells. ( a ) Immunofluorescence of the ER morphology in HUVEC treated with 150 μm of oleate (OA) or palmitate (PA) in combination with DMSO or 2.5 mM 4-PBA. Prior to treatment the cells were transfected with the ER-scarlet probe. Lipid droplets were stained using BODIPY 493/503. The samples were imaged using Zeiss AxioObserver. Scale bar represents 15 μm. (b) Immunoblot analysis of HUVEC treated for 16 h with 150 μm PA in combination with DMSO or 2.5 mM 4-PBA. Cells were lysed in 2x sample buffer and analysed for expression of LC3B, p62, CHOP and Grp78. GAPDH immunblot was used as a loading control. (c) Quantification of immunoblot results from (b). 20 images per condition were analyzed in total in 3 independent experiments. (d) Immunoflourescence analysis of CHOP and p62 expression in HUVEC. The cells were treated as in ( b ), fixed and stained with anti-CHOP and anti-p62 antibodies and DAPI. Scale bar represents 50 μm. (e) Quantification of immunofluorescence analysis depicted in ( d ). For CHOP quantification percentage of CHOP positive cells is displayed and for p62 mean fluorescence intensity (MFI) normalized to control sample. 5 images per condition were analyzed in each of 3 independent experiments. (f) Immunofluorescence analysis of CHOP and p62 expression in HMVEC. Sample preparation and imaging was performed as in ( d ). (g) Quantification of CHOP and p62 staining in HMVEC depicted in ( f ) was performed as described for HUVEC in ( e ). ( i ) FITC labelled dextran leakage assay was performed in HUVEC. Cells were seeded on Transwell inserts and treated for 16 h with BSA + DMSO as a control, 150 µM PA + DMSO or 150 µM PA + 2.5 mM 4-PBA, 150 µM OA or 10 ng/ml TNFa as positive control. The leakage of FITC-labelled 75 kDa Dextran into lower compartment was measured by reading of mean fluorescence intensity at 488 nm using the microplate reader. 3 technical replicates were measured for each condition in 3 independent experiments. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Human Lung Microvascular Endothelial Cells (HMVEC) were obtained from Sciencell Research Laboratories (#sc-3000) and cultured in Endothelial Cell Medium (ECM)(#sc-1001).

Techniques: Inhibition, Immunofluorescence, Transfection, Staining, Western Blot, Expressing, Control, Fluorescence, Sample Prep, Imaging, Positive Control, Software, Comparison, Standard Deviation