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).

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, H₂O₂ 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).

Generation of in vivo knockdown models of CBS and CGL, regulators of transsulfuration pathway involved in Hcy catabolism. (A) Simplified diagram showing the enzymes and metabolites of transsulfuration pathway (B) Representative western blot of 2 dpf embryos lysates showing that compared with SC MO injected embryos, CBS protein level is downregulated in CBS MO injected embryos. Expression of CGL, the other enzyme of the same pathway, remained unaltered. As a loading control β-actin was used. Corresponding bar plot showing fold change in protein expression (normalized to β-actin) as determined by densitometric analysis of the protein band. (C) Bar plot revealing no statistically significant difference in the viability of scrambled control and Hyperhomocysteinemic CBS MO embryos. (D) Bright field images exhibiting no apparent gross morphological defect in CBS morphants compared with scrambled Mo injected embryos of 2 dpf. Scale bar, 0.25 mm. (E) Representative western blot of embryo lysates at 2 dpf showing reduced protein level of CGL in the embryos injected with CGL MO. Expression of CBS, the upstream protein of the same pathway, remained unaltered. β-actin was used as a loading control. Corresponding bar diagram showing densitometric analysis of the fold change in protein expression (normalized to β-actin). (F) In comparison to SC MO injected embryos, viability of CGL MO injected embryos were not altered significantly. (G) Representative bright field images showing absence of any gross morphological defect in CGL MO injected embryos compared with SC MO injected ones at 2 dpf. Scale bar, 0.25 mm. Data are shown as Mean ± SEM with n ≥ 3. ** p ≤ 0.01, **** p ≤ 0.0001 and ns is non-significant (p > 0.05).

Sub-lethal HHcy causes vascular abnormality in vivo without suppressing VEGF/VEGFR transcripts and ROS level change. (A) Upper panel: schematic illustration of transgenic zebrafish line Tg(fli1a:EGFP;gata1a:DsRed) showing anatomical position (rectangle) of CVP and ISV forming area in 36 hpf and 48 hpf embryos respectively. Left panel: representative images revealing impairment in sprouting angiogenesis during CVP formation of CBS morphants. Arrows are depicting CVP sprouts. Scale bar, 0.05 mm. Right panel: representative confocal images showing abnormal ISV formation in CBS morphants compared with SC MO injected embryos. Arrows indicating incomplete, irregular, and abnormally branched ISVs. Scale bar, 50 μm. (B,C) Bar plots respectively showing CVP sprout number and percentage of normal ISV are significantly reduced in CBS morphants. (D) Bar diagram depicting percentage of embryos with angiogenic abnormality is significantly higher in CBS MO injected embryos compared with SC MO injected ones. (E) Representative fluorescent images showing no observable difference in the vascular anatomy of SC MO and CGL MO injected embryos at 2 dpf. Scale bar, 0.2 mm. (F) Bar plot depicting no significant change in percentage of embryos with observable angiogenic abnormality in CGL morphants compared with SC MO injected ones. (G) Bars showing mRNA expression levels of canonical VEGF signaling markers in CBS MO and SC MO injected embryos at 2 dpf. 18S was used as internal control. (H) Representative images depicting location of oxidative stress in 2 dpf zebrafish embryos, detected using general ROS indicator CM-H2DCFDA. White arrows showing drastic increase in ROS levels in the pericardial area of CBS morphants and H₂O₂ treated embryos. White boxes of the tail and regions representing sites of previously observed CVP and ISV angiogenesis, respectively. Scale bar, 0.25 mm. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, *** p ≤ 0.001 and ns is non-significant (p > 0.05).

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).

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).

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.

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.