Use of a Mouse Model and Human Umbilical Vein Endothelial Cells to Investigate the Effect of Arsenic Exposure on Vascular Endothelial Function and the Associated Role of Calpains

Abstract

Background: Arsenic (As) is a well-known environmental contaminant. Chronic exposure to As is known to increase the risk of cardiovascular diseases, including atherosclerosis, hypertension, diabetes, and stroke. However, the detailed mechanisms by which As causes vascular dysfunction involving endothelial integrity and permeability is unclear.

Objectives: Our goal was to investigate how exposure to As leads to endothelial dysfunction.

Methods: Arsenic trioxide (ATO) was used to investigate the effects and mechanisms by which exposure to As leads to endothelial dysfunction using a mouse model and cultured endothelial cell monolayers.

Results: Compared with the controls, mice exposed chronically to As (10 ppb in drinking water supplied by ATO) exhibited greater vascular permeability to Evans blue dye and fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA). In addition, endothelial monolayers treated with ATO ([Formula: see text] As) exhibited greater intracellular gaps and permeability to low-density lipoprotein or transmigrating THP-1 cells. Furthermore, activity and protein levels of calpain-1 (CAPN-1) were significantly higher in aortas and human umbilical vein endothelial cells (HUVECs) treated with ATO. These results were consistent with effects of ATO treatment and included a rapid increase of intracellular calcium ([Formula: see text]) and higher levels of CAPN-1 in the plasma membrane. Endothelial cell dysfunction and the proteolytic disorganization of vascular endothelial cadherin (VE-cadherin) in HUVECs in response to ATO were partially mitigated by treatment with a CAPN-1 inhibitor (ALLM) but not a CAPN-2 inhibitor (Z-LLY-FMK).

Conclusions: This study found that in mice and HUVEC models, exposure to ATO led to CAPN-1 activation by increasing [Formula: see text] and CAPN-1 translocation to the plasma membrane. The study also suggested that inhibitor treatment may have a role in preventing the vascular endothelial dysfunction associated with As exposure. The findings presented herein suggest that As-induced endothelial dysfunction involves the hyperactivation of the CAPN proteolytic system. https://doi.org/10.1289/EHP4538.

Figure 1.

Vascular permeability in mice treated with $10\text{\hspace{0.17em} ppb}$ of arsenic (As). Mice were randomly assigned to four groups ($n=30$ per group) and exposed to 10 ppb As in drinking water supplied by arsenic trioxide (ATO), arsenic pentoxide (arsenate), or roxarsone or maintained on tap water for 4 weeks. Vascular permeability in mice was evaluated by intravenous injection with (A–C) Evans blue or (D–E) fluorescein isothiocyanate–labeled bovine serum albumin (FITC-BSA). Representative images of (A) mice or (B) aortas are shown. The arrows indicate blue coloration. Scale bar: $1\mathrm{cm}$. (C) Evans blue dye in aortas was extracted and measured ($n=3$ per group). (D) Representative images of FITC-BSA in aortic sections are shown (scale bar: $400\mathrm{\mu }\mathrm{m}$ for top images and $100\mathrm{\mu }\mathrm{m}$ for bottom images). (E) Relative fluorescence intensity of FITC-BSA in aortic sections was quantified by ImageJ (three sections per mouse; $n=3$ mice per group). The data are presented as the $\text{mean}\pm \text{standard deviation}$ (SD). ***$p<0.001$ compared with control using one-way ANOVA and Tukey post-test.

Figure 2.

Dose effects of arsenic trioxide (ATO) on endothelial cells. (A) Human umbilical vein endothelial cells (HUVECs) were treated with ATO [$0–4.16\mathrm{\mu }\mathrm{M}$ of arsenic (As)] for 24 h, and cell viability was detected by using the Cell Counting Kit-8 (CCK-8) kit ($n=3$ per group). (B) HUVECs were treated with ATO ($0.13\mathrm{\mu }\mathrm{M}$ As) for the indicated times and concentrations of total arsenic (tAs) was detected ($n=3$ per group). The representative chromatograms of As species, including sodium arsenite [iAs(III)], sodium arsenate [iAs(V)], monomethylarsenic disodium [MMA(V)], and dimethylarsenic acid [DMA(V)], in (C) HUVECs or (E) medium after exposure to indicated doses of ATO for 24 h are shown. The concentrations of As species in (D) HUVECs or (F) medium after a 24-h ATO treatment ($0.13\mathrm{\mu }\mathrm{M}$ As) are shown ($n=3$ per group). The data are presented as the $\text{mean}\pm \text{standard deviation}$ (SD). Note: ANOVA, analysis of variance. *$p<0.05$ or ***$p<0.001$ compared with the $0\mathrm{\mu }\mathrm{M}$ ATO-treated group control using one-way ANOVA and Tukey post-test.

Figure 3.

Calpain (CAPN) activity in the aortas of mice exposed to arsenic trioxide (ATO). (A–D) Mice were randomly assigned to four groups ($n=30$ per group) and exposed to 10 ppb of arsenic (As) in drinking water supplied by ATO, arsenic pentoxide (arsenate), or roxarsone or maintained on tap water for 4 weeks. (A) CAPN activity in aortas was detected ($n=3$ per group). (B) Protein levels of targets in aortas were analyzed by western blotting, and (C) relative protein levels normalized to $\mathrm{\beta }\text{- actin}$ are shown ($n=3$ per group). (D) Relative mRNA levels of targets in aortas were quantified by qPCR ($n=3$ per group). (E–I) Mice were randomly divided into two groups ($n=20$ per group) and maintained on tap water with or without ATO supplementation ($10\text{\hspace{0.17em} ppb}$ As) for 1 week. (E) Mice were euthanized at indicated times and CAPN activity in aortas was detected ($n=3$ per group). (F–I) Mice were injected intravenously with t-BOC-Leu-Met ($20\mathrm{\mu }\mathrm{M}$). Representative images of active CAPNs (pseudocolored in green) in (F) aortas (scale bars: $1\mathrm{cm}$) or (H) aortic sections (scale bars: $400\mathrm{\mu }\mathrm{m}$ for top images and $100\mathrm{\mu }\mathrm{m}$ for bottom images) are shown. Relative fluorescence intensity in (G) aortas ($n=3$ per group) and (I) aortic sections (three sections per animal; $n=3$ animals per group) were quantified by ImageJ. The data are presented as the $\text{mean}\pm \text{standard deviation}$ (SD). Note: ANOVA, analysis of variance; CAST, calpastatin; RFU, relative fluorescence unit; qPCR, quantitative polymerase chain reaction. **$p<0.01$ or ***$p<0.001$ compared with control using one-way ANOVA and Tukey post-test.

Figure 4.

Calpain (CAPN) activity in human umbilical vein endothelial cells (HUVECs) treated with arsenic trioxide (ATO). (A) HUVECs were treated with ATO [$0.13\mathrm{\mu }\mathrm{M}$ of arsenic (As)] for 24 h, and CAPN activity was detected ($n=3$ per group). HUVECs were treated with ATO ($0.13\mathrm{\mu }\mathrm{M}$ As) for (B) 1 h or (C) 0.5 h in the pretreatment with/without BAPTA-AM ($20\mathrm{\mu }\mathrm{M}$, 2 h). (B) Intracellular calcium concentration ($\left[{\mathrm{Ca}}^{2+}\right]\mathrm{i}$) was measured by using Fura-2 AM and $\left[{\mathrm{Ca}}^{2+}\right]\mathrm{i}$ was calculated ($n=3$ per group; arrow indicates ATO addition). (C) CAPN activity was detected ($n=3$ per group). PBS and DMSO were served as solvent controls. (D) Gene expression of CAPNs or calpastatin (CAST) in HUVECs treated with ATO ($0.13\mathrm{\mu }\mathrm{M}$ As, 12 h) was assessed by qPCR ($n=3$ per group). (E) Protein levels in HUVECs treated with ATO ($0.13\mathrm{\mu }\mathrm{M}$ As) for the indicated times were assessed by western blotting and (F) relative protein levels normalized to $\mathrm{\beta }\text{- actin}$ are shown ($n=3$ per group). The data are presented as the $\text{mean}\pm \text{standard deviation}$ (SD). Note: ANOVA, analysis of variance; DMSO, dimethylsulfoxide; PBS, phosphate-buffered saline; RFU, relative fluorescence unit; qPCR, quantitative polymerase chain reaction. **$p<0.01$ or ***$p<0.001$ compared with their own control using one-way ANOVA and Tukey post-test.

Figure 5.

Protein levels and activity of calpains (CAPNs) in membrane fraction of human umbilical vein endothelial cells (HUVECs) treated with arsenic trioxide (ATO). HUVECs were treated with ATO [$0.13\mathrm{\mu }\mathrm{M}$ of arsenic (As)] for the indicated times. (A) Proteins levels of calpastatin (CAST) and CAPNs in cytosolic and membrane fractions were analyzed by western blotting. Relative protein levels in (B) cytosolic and (C) membrane fractions normalized to $\mathrm{\beta }\text{- actin}$ or ${\mathrm{Na}}^{+}{/\mathrm{K}}^{+}\text{- ATPase}$ are shown ($n=3$ per group). (D) CAPN activity in different fractions was detected ($n=3$ per group). (E) CAPN-1 in cells treated with ATO ($0.13\mathrm{\mu }\mathrm{M}$ As) for the indicated times was detected a using rabbit polyclonal antibody followed by goat anti-rabbit antibody (Cy3-conjuated) and nuclei were counterstained with DAPI. Representative images are shown (scale bars: $20\mathrm{\mu }\mathrm{m}$) and arrows indicate foci of CAPN-1 in membrane. The data are presented as the $\text{mean}\pm \text{standard deviation}$ (SD). Note: ANOVA, analysis of variance; RFU, relative fluorescence unit. *$p<0.05$, **$p<0.01$, or ***$p<0.001$ compared with control (0 h) using one-way ANOVA and Tukey post-test.

Figure 6.

Effect of the CAPN-1 inhibitor, ALLM, on endothelial permeability in monolayers following arsenic trioxide (ATO)–treatment. Human umbilical vein endothelial cells (HUEVCs) were seed onto (A–C) glass bottom dishes or (D–G) Transwell^® permeable supports to establish monolayers and then treated with ATO [$0.13\mathrm{\mu }\mathrm{M}$ of arsenic (As)] in the presence/absence of ALLM ($10\mathrm{\mu }\mathrm{M}$) or the CAPN-2 inhibitor, Z-LLY-FMK, ($4\mathrm{\mu }\mathrm{M}$) for 24 h. (A–B) Monolayers were stained with DiI and Hoechst. Representative (A) fluorescence images and (B) fluorescence images merged with the corresponding brightfield images are shown (scale bars: $20\mathrm{\mu }\mathrm{m}$; arrows indicate intracellular gaps). (C) Intracellular gaps were quantified as the percent area of monolayers uncovered by cells (three images per dish, $n=3$ dishes per group). (D) Transendothelial electrical resistance (TEER) was detected at indicated times ($n=3$ per group). Treated monolayers were co-cultured with (E) DiI-ox-LDL ($30\mathrm{\mu }\mathrm{g}/\mathrm{mL}$, 24 h) or (F–G) activated human monocytic cell line (THP-1) cells ($1\times {10}^4$ per well, 6 h). (E) Relative fluorescence intensity of DiI-ox-LDL in the outer chamber was detected at indicated times ($n=3$ per group). (F) Migrated THP-1 cells in membranes were stained with calcein-AM, DiI, and Hoechst, and representative images are shown (scale bars: $400\mathrm{\mu }\mathrm{m}$). (G) The total numbers of migrated cells were counted (three images per membrane, $n=3$ membranes per group). The data are presented as the $\text{mean}\pm \text{standard deviation}$ (SD). Note: ANOVA, analysis of variance. Different letters indicate significant difference between groups ($p<0.05$) as determined by one-way ANOVA and Tukey post-test.

Figure 7.

Effect of the CAPN-1 inhibitor, ALLM, on proteolysis of vascular endothelial cadherin (VE-cadherin) following arsenic trioxide (ATO)–treatment in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with ATO [$0.13\mathrm{\mu }\mathrm{M}$ arsenic (As)] in the presence/absence of ALLM ($10\mathrm{\mu }\mathrm{M}$) or the CAPN-2 inhibitor (Z-LLY-FMK) ($4\mathrm{\mu }\mathrm{M}$) for 24 h. (A) Proteolysis of VE-cadherin was analyzed by western blot. (B) The graph represents the ratio of cleaved/uncleaved ($90/135\text{\hspace{0.17em} kDa}$) bands of VE-cadherin ($n=3$ per group). (C) VE-cadherin in cells was detected using rabbit monoclonal antibody followed by goat anti-rabbit antibody (Cy3-conjuated). Nuclei were counterstained with DAPI. Representative images are shown (scale bars: $20\mathrm{\mu }\mathrm{m}$), and arrow indicates proteolytic disorganization of VE-cadherin. The data are presented as the $\text{mean}\pm \text{standard deviation}$ (SD). Note: ANOVA, analysis of variance. Different letters indicate significant difference between groups ($p<0.05$) as determined by one-way ANOVA and Tukey post-test.