Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethally increased Hcy causes endothelial dysfunction. ( A ) Bar graph represents percentage of cell death in HUVEC/TERT2 cells treated with increasing concentration of Hcy for 24 h. A sub-lethal concentration of 2 mM (24 h) was chosen for all the subsequent experiments. ( B ) HPLC mediated quantification of intracellular Hcy concentration in HUVEC/TERT2 cells revealing induction of moderate Hyperhomocysteinemic condition post 2 mM Hcy treatment for 24 h. ( C ) Representative images of tube formation assay showing functional abnormality in Hcy treated HUVEC/TERT2 cells compared with untreated cells. Scale bar, 250 μm. ( D , E ) Respective quantifications showing total tube length and number of branches formed during tube formation assay are drastically reduced upon 2 mM Hcy treatment for 24 h. Data are shown as Mean ± SEM with n ≥ 3. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Concentration Assay, Tube Formation Assay, Functional Assay

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethal HHcy reduces endothelial migration and proliferation without suppressing VEGF/VEGFR transcripts and ROS level change. ( A ) Scratch wound assay in presence of Mitomycin C showing less migrated endothelial cells at 24 h post 2 mM Hcy treatment. ( B ) Quantification of migrated cells revealing that endothelial migration is significantly reduced in Hcy treated cells compared with control cells. ( C ) Bar plot of BrdU cell proliferation assay indicating that 2 mM Hcy treatment for 24 h causes proliferation defect in endothelial cells. ( D ) Representing scratch wound assay images depicting that as opposed to Hcy treatment, a similar concentration of Cys did not affect migration of endothelial cells when compared with untreated cells at 24 h. ( E ) Bar plot of measurement of migrated cells in scratch wound assay showing that contrary to Hcy treated cells, fold change in migrated cells is not altered upon 2 mM Cys treatment for 24 h compared with control cells. ( F ) BrdU cell proliferation assay revealing that unlike Hcy treated cells, 2 mM Cys treatment for 24 h did not influence endothelial proliferation. ( G ) Bars showing that compared with untreated cells, exposure to sub-lethal Hcy caused upregulation of mRNA levels of canonical VEGF signaling markers. 18S was used as internal control. ( H ) Bar graph showing that sub-lethal Hcy treatment does not induce ROS production in endothelial cells as determined by the fluorescent probe CM-H2DCFDA. For positive control, H 2 O 2 was used. ( I ) Representative western blots showing protein levels of major antioxidant markers GPX1 and SOD1. Corresponding bar graphs showing densitometric analysis of the protein bands which suggest a non-significant but slight trend of upregulation of both the proteins in 2 mM Hcy (24 h) treated cells. As a loading control β-actin was used. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Migration, Scratch Wound Assay Assay, Control, BrdU Cell Proliferation Assay, Concentration Assay, Positive Control, Western Blot

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethal HHcy-induced adaptive UPR controls endothelial migration defect. ( A ) Representative western blots showing protein levels of UPR markers GRP78, IRE1p and ATF4 are upregulated upon 2 mM Hcy treatment for 24 h. Terminal UPR marker CHOP remained unaltered post-sub-lethal Hcy treatment. As a loading control β-actin was used. Corresponding bar graph showing densitometric analysis (normalized to β-actin) of the blots. ( B , C ) Respective quantifications of scratch wound assay at 24 h revealing that chemical chaperone 4-PBA (1 mM) and TUDCA (1 mM) can significantly improve sub-lethal HHcy-induced endothelial migration defect. ( D ) Representative confocal images of rhodamine-phalloidin stained endothelial cells showing that sub-lethal Hcy-induced abnormally elongated cell morphology as well as actin stress fiber (white arrows) disappearance are rescued by 4-PBA pre-treatment. Scale bar, 5 μm. ( E ) ImageJ based analysis demonstrating that 4-PBA pre-treatment reversed the aberrant reduction in cellular aspect ratio (major axis/minor axis), induced by 24 h treatment of 2 mM Hcy. ( F ) Quantification by ImageJ suggesting that exposure to sub-lethal Hcy significantly decreased the surface area of endothelial cells which was rescued by 4-PBA. ( G ) Bar plot showing no beneficial effect of chemical chaperone TUDCA on impairment of endothelial proliferation caused by sub-lethal Hcy treatment. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Migration, Western Blot, Marker, Control, Scratch Wound Assay Assay, Staining

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethal HHcy linked malfunctional ETC impairs mitochondrial respiration of endothelial cells. ( A ) OCR curves showing drastic reduction in endothelial mitochondrial respiration upon 2 mM Hcy treatment for 24 h as measured by an extracellular flux analyzer. ( B – D ) Respective bar graphs demonstrating that in comparison to untreated cells, there is significant decrease in basal respiration, ATP production and maximal respiration of endothelial cells with sub-lethal HHcy. ( E ) OCR curves showing no restoration of sub-lethal HHcy-induced mitochondrial respiration defect in presence of TUDCA. ( F – H ) Bar plots respectively showing no significant improvement in the reduction in basal respiration, ATP production and maximal respiration upon TUDCA pre-treatment, compared with sub-lethal Hcy treated endothelial cells. ( I ) Targeted metabolomics mediated quantification exhibiting significant elevation of metabolites of TCA cycle in sub-lethal Hcy treated endothelial cells compared with untreated control cells. AUC, area under the curve. ( J ) Bar plot showing that sub-lethal HHcy in endothelial cells causes significant reduction in enzymatic activity of COX, the terminal electron acceptor of ETC. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Comparison, Control, Activity Assay

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Glycolysis is elevated upon induction of sub-lethal HHcy in endothelial cells. ( A ) ECAR curves showing drastically upregulated glycolysis of endothelial cells treated by 2 mM Hcy for 24 h as measured by an extracellular flux analyzer. ( B – D ) Respective bar graphs revealing that in comparison to untreated cells, there is a significant enhancement of glycolysis, glycolytic reserve, and glycolytic capacity of sub-lethal Hcy treated endothelial cells. ( E ) Bar graph of targeted metabolomics showing metabolic intermediates of glycolysis are elevated in endothelial cells with sub-lethal HHcy. AUC, area under the curve. ( F ) Glucose uptake assay using fluorescence analog 2-NBDG showing that in comparison to control cells, consumption of extracellular glucose is higher in sub-lethal Hcy treated endothelial cells. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Comparison, Fluorescence, Control

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Mechanistic features of pathologically relevant Hcy exposure are conserved in adult endothelial cells. ( A ) Schematic diagram showing experimental model and treatment condition used for microarray profiling of GSE175735 from GEO database, previously published by M Jan et al. ( B ) Heatmap illustrating the trend of downregulation in differentially expressed genes (DEGs) of cell cycle and cellular migration compared between control and Hcy treated (0.5 mM, 48 h) groups. ( C – E ) Respective heatmaps showing pathway specific expression of angiogenesis and antioxidant response genes, UPR and metabolism (TCA cycle and glycolysis), in the same Hcy treated and untreated control cell groups. Statistically significant ( p -value < 0.05) genes were considered to obtain DEGs. Respective color legends representing Z-score ((observed value–mean)/standard deviation) values. Three sets of samples each from control and Hcy treated cells were used for analysis.

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Microarray, Migration, Control, Expressing, Standard Deviation

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 1 High glucose and TNFα induce a profound gene expression and phenotypic change. a HUVECs were treated in biological duplicates as Day 0: 25 mM mannitol as normal glucose and osmolarity control (NM); Day 3: combined treatment consisting 25 mM D-glucose and 5 ng/mL TNFα (H + T) for 3 days; and Day 7: H + T treatment for 7 days. Each group of treated cells was subjected to single-cell RNA-seq (scRNA-seq), Hi-C, and iMARGI assays. b t-SNE plot of scRNA-seq (4000–15,000 cells per sample) showed clear separation by treatment condition into three distinct clusters. c Principal component analysis of scRNA-seq data: single cells are plotted in the first two PC space and are labeled in red (Day 0, i.e., NM), green (Day 3, i.e., 3-day H + T treatment), and blue (Day 7, i.e., 7-day H + T treatment). d Expression heatmap (z-scaled) of top DE genes in single ECs grouped into functional pathways. Cells were ordered by increasing SERPINE1 expression (per each sample separately) and binned per 100 cells for the analysis. A total of 269 bins in Day 0, 177 bins in Day 3, and 148 bins in Day 7. e t-SNE plots of the expression level of selected genes in each single cell across the time course. The RNA levels are represented by log-normalized unique molecular identifier counts. f mRNA levels of eNOS and α-SMA in NM vs. H + T-treated HUVECs and cells untreated (NT) or treated with TGF-β (10 ng/mL) and IL-1β (5 ng/mL; T + I) for 3 or 7 days. The respective control was set as 1. Relative eNOS level: data represent mean ± SEM from five independent experiments; relative α-SMA level in H + T treatment: data represent mean ± SEM from seven independent experiments; relative α-SMA level in T + I treatment: data represent mean ± SEM from four independent experiments. *P = 0.0067, 0.0087, 0.0057, and 0.0017 from left to right based on ANOVA with Bonferroni as post hoc test. g Cell morphology under bright field (BF), immunofluorescent staining of α-SMA, and VE-cadherin (VE-cad), phalloidin staining of cytoskeleton, and DRAQ5 (DRAQ) staining of the nuclei. Representative images from five independent experiments are shown. Scale bar of BF = 100 µm; scale bars of (immuno)fluorescent staining = 50 µm. Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Gene Expression, Control, RNA Sequencing, Hi-C, Labeling, Expressing, Functional Assay, Staining

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 2 Overview of time-course Hi-C and iMARGI data. a, b Proportions of intrachromosomal (yellow) and interchromosomal read pairs (blue) in Hi-C (a) and iMARGI data (b) at the three time points (columns). c An example of interchromosomal iMARGI read pairs mapped to chromosome 2 near LINC00607 (left) and chromosome 7 near SERPINE1 (right). The RNA end (pink) and the DNA end (green) of each read pair is linked by a horizontal line. d Examples of overlapping iMARGI read pairs on a contact matrix from the RNA end (rows) to the DNA end (columns) with SEs (marked in light blue and in SE tracks) in control (Day 0) ECs. Genome region: chr1:75,000,0000–chr1:125,000,000. Resolution = 200 kb. e Proportions of iMARGI read pairs with the RNA ends (pink) or the DNA ends (green) in Day 0 (Ctrl) and Days 3 and 7 (H + T) ECs mapped to HUVEC SEs. Dotted line: the relative size of HUVEC SEs compared to the size of the genome. Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Hi-C, Control

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 4 Inhibition of LINC00607 attenuates SERPINE1 induction and EC dysfunction. a Illustration of LINC00607 genomic locus and gene structure and LNA GapmeRs targeting of LINC00607 RNA. b HUVECs were transfected with scramble (scr) or two LNAs targeting LINC00607. LINC00607 RNA and SERPINE1 mRNA levels were quantified. Data represent mean ± SEM from seven independent experiments. c qPCR of LINC00607 in subcellular fractionations of HUVECs transfected with scr, LNA1, or LNA2. Data represent mean ± SEM from four independent experiments. d RNA-seq was performed with cells transfected as in b in biological replicates. Heatmap is plotted based on z-scaled log-transformed gene expression levels. e, f ECs transfected with scr or LNA1 and then treated by H + T were used in e monocyte adhesion assay and in f SA-β-gal assay. In e, the number of attached monocytes to ECs were quantified. Data represent mean ± SEM from eight independent experiments performed with peripheral blood-derived monocytes from four individual donors. Representative images show the attachment of fluorescently labeled peripheral blood-derived monocytes to ECs for experiments performed with monocytes from three different donors. In f, ECs with positive β-gal staining were quantified. The positively stained cell number in scr control was set to 100 (%). f Data represent mean ± SEM from four independent experiments respectively. Scale bar = 200 μm. * denotes p = 0.0289, 0.0013, 0.001, 0.0001, 0.0025, and 0.024 from left to right (in b) and p = 0.0023, 0.0079, and 0.0249 from left to right (in c) between indicated groups based on ANOVA followed by Bonferroni post hoc test (in b and c); p = 0.0039 (in e) and 0.0014 (in f), as compared to scr group based on two-tailed paired t test (in e and f). Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Inhibition, Transfection, RNA Sequencing, Transformation Assay, Gene Expression, Cell Adhesion Assay, Derivative Assay, Labeling, Staining, Control, Two Tailed Test