primary human lung microvascular endothelial cells (ecs, cc-2527)  (Lonza)

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: The FASEB Journal

Article Title: Primary endothelial cell–specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia

doi: 10.1096/fj.201802650RR

Figure Lengend Snippet: The hypoxic switch between HIF-1α and HIF-2α constitutes a universal mechanism of human endothelium adaptation to prolonged hypoxia. A) The mathematic representation of HIF-1α/HIF-2α switch in human ECs. The changes in HIF-1α (obtained from vascular or microvascular ECs) and HIF-2α levels (from all ECs tested) during hypoxia time course were analyzed using the logarithmic normal 3 parameter function (using 200 iterations, P < 0.005). The dashed lines represent predicted normoxic HIF levels. The error bars represent sd. B) The general model of endothelial HIF-1α/HIF-2α proposed based on the mathematical modeling. Max., maximum.

Article Snippet: Primary human cardiac microvascular ECs (HMVEC-Cs), primary human dermal microvascular ECs (HMVEC-Ds), primary human lung microvascular ECs (HMVEC-Ls), and primary human uterine microvascular ECs (UtMVECs) were purchased from Lonza and cultured in microvascular EGM-2 (EGM-2MV) medium (Lonza).

Techniques:

Journal: Cells

Article Title: MicroRNA-Regulated Rickettsial Invasion into Host Endothelium via Fibroblast Growth Factor 2 and Its Receptor FGFR1

doi: 10.3390/cells7120240

Figure Lengend Snippet: Expression levels of miR-424 and miR-503 in different types of endothelial cells infected with R. conorii . HMECs ( A ), HLMECs ( B ), and HCECs ( C ) were infected with R. conorii (Rc) for various time periods up to 6 h. RNA was extracted and qRT-PCR assays were performed to measure the expression of miR-424 and miR-503. The data was normalized to 18S RNA, and relative expression was calculated by the ΔΔ Ct method. The results are presented as the mean ± standard error (SE) of three independent experiments. The asterisk indicates statistically significant change ( p ≤ 0.01). HMECs: human dermal microvascular endothelial cells; HLMECs: human lung microvascular endothelial cells; HCECs: human cerebral microvascular endothelial cells; Con: control.

Article Snippet: Primary human lung microvascular ECs (HLMECs) were purchased from Lonza and maintained in culture according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Quantitative RT-PCR, Control

Journal: Cells

Article Title: MicroRNA-Regulated Rickettsial Invasion into Host Endothelium via Fibroblast Growth Factor 2 and Its Receptor FGFR1

doi: 10.3390/cells7120240

Figure Lengend Snippet: Expression levels of FGF2 and FGFR1 mRNA in R. conorii -infected host endothelial cells. HMECs ( A ), HLMECs ( B ), and HCECs ( C ) were infected with R. conorii for various time periods up to 6 h. RNA was extracted, and qRT-PCR assays were performed to measure the FGF2 and FGFR1 expression. The data was normalized to 18S rRNA and relative expression was calculated by the ΔΔ Ct method. The results are presented as the mean ± SE of three independent experiments. The asterisk indicates statistically significant change ( p ≤ 0.01).

Article Snippet: Primary human lung microvascular ECs (HLMECs) were purchased from Lonza and maintained in culture according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Quantitative RT-PCR