Biology of Reproduction, 2017, 96(1), 199–210 doi:10.1095/biolreprod.116.142604 Research Article Advance Access Publication Date: 22 December 2016

Research Article

Vitamin D suppresses oxidative stress-induced microparticle release by human umbilical vein endothelial cells Xiuyue Jia1,2 , Jie Xu1 , Yang Gu1 , Xin Gu3 , Weimin Li2 and Yuping Wang1,∗ 1

Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, USA; 2 Department of Cardiology, The First Affiliated Hospital Harbin Medical University, Harbin, China and 3 Department of Pathology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, USA ∗

Correspondence: Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center-Shreveport, 1501 Kings Highway, Shreveport, LA 71130, USA. Tel: (318) 675-5379; E-mail: [email protected] Grant support: This study was supported in part by grant from National Institutes of Health (NIH), Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) R21HD076289 to Yuping Wang. The work was presented at the 63rd Annual Meeting of the Society for Reproductive Investigation, Montreal, Canada, March 16–19, 2016. Received 10 June 2016; Revised 1 November 2016; Accepted 15 November 2016

Abstract Endothelial microparticle (MP) release was increased in numerous cardiovascular diseases including preeclampsia. Oxidative stress is a potent inducer of endothelial dysfunction. In this study, we aimed to investigate if vitamin D could protect endothelial cells (ECs) from MP release induced by oxidative stress. Endothelial cell (from human umbilical vein) oxidative stress was induced by cultivation of cells under lowered oxygen condition (2%O2 ) for 48 h and cells cultured under standard condition (21%O2 ) served as control. 1,25(OH)2 D3 was used as bioactive vitamin D. Using annexinV as a marker of released MP assessed by flow cytometry and cytochrome c reduction assay to measure EC superoxide generation, we found that MP release and superoxide generation were significantly increased when cells were cultured under 2%O2 , which could be significantly inhibited by 1,25(OH)2 D3 . To study the potential mechanisms of 1,25(OH)2 D3 protective effects on ECs, EC expression of endothelial nitric oxide synthase (eNOS), p-eNOSSer1177 , p-eNOSThr495 , caveolin-1, extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-AktSer473 , Rho-associated coiled-coil protein kinase 1 (ROCK1), and vitamin D receptor were determined. Microparticle expression of eNOS and caveolin-1 were also determined. We found that under lowered oxygen condition, 1,25(OH)2 D3 could upregulate EC eNOS, p-eNOSSer1177 , and p-AktSer473 expression, but inhibit cleaved ROCK1 expression. The upregulatory and inhibitory effects induced by 1,25(OH)2 D3 were dose dependent. Strikingly, we also found that oxidative stress-induced decrease in ratio of eNOS and caveolin-1 expression in MP could be attenuated when 1,25(OH)2 D3 was present in culture. These results suggest that upregulation of eNOSSer1177 and AktSer473 phosphorylation and inhibition of ROCK1 cleavage in EC and modulation of eNOS and caveolin-1 expression in MP could be plausible mechanisms of vitamin D protective effects on ECs.

 C The Authors 2016. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For

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Summary Sentence 1,25(OH)2 D3 induced upregulation of eNOSSer1177 and AktSer473 phosphorylation and inhibition of ROCK1 cleavage could be plausible mechanisms of vitamin D protective effects to suppress MP release by ECs. Key words: endothelial cells, microparticles, eNOS, Akt, ROCK, vitamin D.

Introduction Endothelial microparticles (MPs) are complex vesicular structures shed from endothelial cells (ECs) to the circulation. Under physiological condition, endothelial MPs contribute to cellular communication and help to maintain vascular homeostasis [1]. However, activated and/or apoptotic ECs release many more MPs under stress conditions and these MPs have been proven to play significant roles in inflammation, coagulation, and angiogenesis, and contribute to the progression of many vascular diseases [2, 3]. For example, elevated levels of endothelial MPs are found in plasma from patients with cardiovascular diseases including hypertension, insulin resistance, diabetes mellitus, and even abdominal obesity [4]. Increased circulating MPs of endothelial origin are also found to be closely associated with endothelial dysfunction in patients with end-stage renal failure [5]. In addition, recent studies also revealed that circulating endothelial MPs were increased in pregnancy disorders such as in preeclampsia, a hypertensive disorder in human pregnancy [6, 7]. In preeclampsia, elevated endothelial MP levels were found to be positively correlated with umbilical and middle cerebral artery resistance index [6], suggesting the pivotal role of increased endothelial MP production related to vascular dysfunction. Thus, it is widely accepted that elevation of plasma endothelial MP levels particularly reflects cellular injury and appears as both the marker and activator of vascular dysfunction. Bioactive vitamin D including 25-hydroxyvitamin D3 (25(OH)D3 ) and 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) is a group of fat-soluble secosteroids responsible for regulating calcium and phosphorus absorption and maintaining healthy bones in humans. Emerging evidence has shown that vitamin D is not only one of the most important nutrients for bone health, but it also exerts beneficial effects on multiple organs and systems through modulating adaptive immunity, vascular inflammation, and endothelial function. Studies have also revealed that vitamin D reduces the risk of several chronic diseases including cancer, type 1 diabetes, and heart diseases [8]. Although it is still controversial if vitamin D therapy could reduce blood pressure in hypertensive patients, experimental studies have demonstrated novel actions of vitamin D metabolites on cardiomyocytes, ECs, and vascular smooth muscle cells [9]. We previously reported that vitamin D could suppress thromboxane production induced by oxidative stress in placental trophoblasts and vitamin D could also upregulate CuZn-SOD expression in ECs [10, 11]. These findings suggest that vitamin D may exert antioxidative properties. Since increased endothelial MP release plays significant roles in endothelial dysfunction, contributes to cardiovascular diseases, and promotes endothelial inflammatory response during pregnancy, in this study we aimed to investigate if vitamin D could protect ECs to suppress endothelial MP release induced by oxidative stress. Endothelial oxidative stress was induced by incubation of cells under lowered oxygen (2%O2 ) condition. 1,25(OH)2 D3 was used as a bioactive vitamin D. Endothelial nitric oxide synthase (eNOS) and caveolin-1 expression as well as ERK, Akt, and Rho-associated coiled-coil protein kinase 1 (ROCK1)

expression were examined to determine the potential mechanism of vitamin D protective effects on ECs.

Materials and methods Chemicals and reagents 1,25(OH)2 D3 (D1570), cytochrome c (C4186), and superoxide dismutase (S2515) were purchased from Sigma Chemicals (St. Louis, MO, USA). Phorbol 12-myristate 13-acetate (P-1680) was from LC Laboratories (Woburn, MA, USA). Antibodies for vitamin D receptor (VDR) (D-6, sc-13133), eNOS (C-20, sc-654), caveolin-1 (N-20, sc-894), ERK (C-16, sc-93), p-ERK (E-4, sc-7383), and ROCK1 (G6, sc-17794) were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). Antibodies for phospho-eNOS (Ser1177) (C9C3, product# 9570), phospho-eNOS (Thr495) (product# 9574), Akt (C67E7, product# 4691), and phospho-Akt (Ser473) (D9E, product# 4060) were obtained from Cell Signaling Technology (Danvers, MA, USA). β-Actin antibody was from Sigma Chemicals. Annexin V-APC (BD 550474) was obtained from BD Biosciences (San Jose, CA). Type II collagenase was obtained from Worthington Biochemical Corporation (Lakewood, NJ, USA). All the other chemicals and reagents were from Sigma Chemicals unless otherwise noted.

Human umbilical vein endothelial cell isolation and culture Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase digestion from umbilical cord vein of normal term placentas (n = 18) as previously described [12, 13]. Collection of placental umbilical cords for HUVEC isolation was approved by Institutional Review Board for Human Research at Louisiana State University Health Sciences Center-Shreveport, Louisiana. Isolated ECs were incubated with EC growth medium purchased from Lonza (Walkersville, MD, USA). Passage 2–3 cells were used in the experiments. Endothelial cells were grown in six well per plate and incubated in either standard cell culture condition (5%CO2 /95% air) or lowered oxygen condition (2%O2 /5%CO2 /93%N2 ) in the presence or absence of 1,25(OH)2 D3 at 50 nM for 48 h. A concentration of 1,25(OH)2 D3 at 50 nM used was based on our previously published work, in which 1,25(OH)2 D3 at concentrations of 5–100 nM could induce endothelial CuZn-SOD and VDR expression [11]. For lowered oxygen culture condition, the plates were placed in a portable air chamber (Billups-Rothenberg, Del Mar, CA) for 2 days. The chamber was flushed daily with a gas mixed with 2% O2 , 5% CO2 balanced with 93% N2 for 2 min at 10 L/min. The “lowered O2 ” condition represents a range of ambient O2 of 2% ± 0.01. The chamber was housed in a humidified incubator to maintain at 37◦ C. Cells incubated in a standard culture incubator (5%CO2 /95% air) served as control. For dose effect studies, ECs were treated with 1,25(OH)2 D3 at concentrations of 0, 10, 50, and 100 nM and cultured under 2%O2 for 48 h. At the end of incubation, cell culture supernatant was collected for extraction of endothelial MPs and total cellular protein was collected for target protein expression studies.

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Endothelial microparticle isolation and flow cytometry analysis Endothelial MPs were isolated from cell culture supernatant by a two-step centrifugation procedure modified based on previous published work [14]. Briefly, medium samples were first centrifuged at 400 × g for 20 min at 4◦ C to remove cell debris, and then the supernatant was centrifuged again at 20,000 × g for 60 min at 4◦ C. After washing with phosphate buffer saline (PBS), extracted MPs were (1) labeled with annexin-V conjugated with fluorochrome APC (annexin-V APC) for flow cytometry analysis, (2) fixed with 2.5% glutaraldehyde for transmission electron microscope (TEM) examination, or (3) lysed to obtain total MP protein for protein expression study. For flow cytometry analysis, MPs were incubated with 5 μl of annexin-V APC in 100 μl of annexin-V binding buffer (BD Biosciences, product# 556454) containing 10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2 . After 15 min incubation in dark, each sample was then diluted again with 400 μl of annexin-V binding buffer and analyzed by a BD LSR II flow cytometer (BD Biosciences). Megamix-Plus side scatter (SSC) beads from Biocytex (Marseille, France) were used to set the size gate of MP captured. The intensity of annexin-V APC binding was evaluated in APC-fluorescence histogram plot. TruCount tube from Becton Dickinson (San Diego, CA) with a known number of fluorescent beads was used in each sample as an internal standard. Data were analyzed using FlowJo cell analysis software (Tree Star, Ashland, OR, USA). Microparticles count was normalized by total cellular protein per well.

Transmission electron microscopy Isolated MPs were fixed with 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide mixed with 0.8% potassium ferricyanide in 0.1 M, pH 7.35 cacodylate buffer. After dehydration in acetonic series (50%, 70%, 90%, and 100%), MPs were embedded in epoxy resin. Ultrathin sections (90 nm) were cut on a Lecia EM UC6 ultratome and mounted on 200-mesh copper grids. Ultrathin sections were then stained with uranyl acetate-lead citrate solution and examined by a Hitachi H-7650 TEM (Japan).

Superoxide generation assay Endothelial superoxide generation was measured by cytochrome c reduction assay as previously described [15]. Briefly, cells were washed with prewarmed PBS and then treated with either superoxide dismutase or equal volume of water with Hanks Balanced Salt Solution at 37◦ C for 2 min. After adding phorbol myristate acetate and cytochrome c, cells were incubated at 37◦ C for 15 min. Supernatant was then collected by centrifugation and cytochrome c reduction was measured in a double-beam spectrophotometer (Ultrospec 3000, Pharmacia Biotech, Cambridge, England) by scanning the supernatant with wavelength at 530–570 nm. Dismutase-containing supernatant was used as the contrast. The height of the peak at 550 nm represents the absorbance due to superoxide-dependent cytochrome c reduction (Asuperoxide ). The amount of superoxide generation was calculated as follows: o2− (nmol) = 47.7 × Asuperoxide , and normalized by total cellular protein.

Protein expression After 48 h incubation, cells or isolated MPs were lysed with lysis buffer containing 50 mmol/L Tris, 0.5% NP40, 0.5% Triton X100 with protease and phosphatase inhibitors. Protein expression for caveolin-1, eNOS, p-eNOSSer1177 , p-eNOSThr495 , ERK, p-ERK,

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ROCK1, Akt, p-AktSer473 , and VDR in ECs and protein expression for eNOS and caveolin-1 in MPs were determined by western blot. Briefly, an aliquot of 10 μg of total endothelial cellular protein or whole lysate of endothelial MPs isolated per well was subject to electrophoresis (Bio-Rad, Hercules, CA) and then transferred to nitrocellulose membranes. After blocking, the membranes were probed with a specific antibody followed by a matched secondary antibody. An enhanced chemiluminescent detection kit (Amersham Corporation, Arlington Heights, IL) and X-ray film were used to visualize and expose the bound antibody, respectively. Membranes were then stripped and reprobed with different antibodies. After scanning, the density of bands was analyzed by NIH Image J analysis program. β-Actin expression was used to normalize the target endothelial protein expression. Data were presented as mean ± SE from five to six independent experiments.

Statistical analysis A computer software program Prism 5 (GraphPad Software, Inc., La Jolla, CA) was used for statistical analysis. Paired t-test was used to compare cells that were cultured between standard culture condition versus lowered oxygen condition, and between cells treated with and without 1,25(OH)2 D3 . Analysis of variance was used to compare dose responses for 1,25(OH)2 D3 in cells cultured under lowered oxygen culture condition. The Student-Newman-Keuls test was used as a post hoc test. A probability level less than 0.05 was considered statistically significant.

Results 1,25(OH)2 D3 reduces microparticle release by endothelial cells cultured under lowered oxygen condition To study protective effect of 1,25(OH)2 D3 on ECs, we first determined if 1,25(OH)2 D3 could suppress MP release by ECs under oxidative stress challenge. As shown in Figure 1A, we found that cells cultured under lowered oxygen (2%O2 ) released significantly more MPs than cells that were cultured under standard culture condition (21%O2 ), P < 0.05. Although there was no significant difference for annexin-V positive MP release in cells cultured under standard culture condition with or without 1,25(OH)2 D3 , the increased MP release by cells cultured under lowered oxygen was significantly reduced when 1,25(OH)2 D3 was present in culture, P < 0.05.

1,25(OH)2 D3 inhibits superoxide generation induced by oxidative stress To further assess the potential antioxidative effects of 1,25(OH)2 D3 , we examined superoxide generation in ECs cultured under lowered oxygen or standard oxygen conditions in the presence or absence of 1,25(OH)2 D3 . Our results showed that cells cultured under lowered oxygen condition produced significantly more superoxide than cells incubated under standard culture condition, P < 0.01 (Figure 1B). Consistent with MP release, superoxide generation was not different in cells incubated under standard culture condition with or without 1,25(OH)2 D3 treatment, but increased superoxide generation by cells cultured under lowered oxygen condition was significantly reduced when cells were treated with 1,25(OH)2 D3 , P < 0.05. These results suggest that 1,25(OH)2 D3 could protect ECs from oxidative stress challenge by inhibiting superoxide generation.

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Figure 1. Effects of 1,25(OH)2 D3 on MP release and superoxide generation by ECs cultured under 2%O2 and 21%O2 . A) Annexin-V positive MP release from ECs was assessed by flow cytometry and the count was normalized by total cellular protein. Annexin-V positive MP release was significantly increased in cells cultured under 2%O2 vs. 21%O2 , ∗ P < 0.05, which could be suppressed when 1,25(OH)2 D3 was present in culture, # P < 0.05. B) Superoxide generation by ECs was assessed by cytochrome c reduction assay and normalized by total cellular protein. Superoxide generation was significantly increased when cells were cultured under 2%O2 vs. 21%O2 , ∗ ∗ P < 0.01. Increased superoxide generation could be attenuated by 1,25(OH)2 D3 , # P < 0.05. ∗ P < 0.05 and ∗ ∗ P < 0.01: untreated cells cultured under 2%O2 vs. untreated cells cultured under 21%O2 ; # P < 0.05: 1,25(OH)2 D3 treated cells cultured under 2%O2 vs. untreated cells cultured under 2%O2 , respectively. Data are means ± SE from five to six independent experiments. C) A representative image of isolated EC MPs examined by TEM, showing that isolated MPs are intact and the size within the MP limitation between 0.1 and 1.0 μm in diameter, bar = 200 μm. D) Protein expression for eNOS and caveolin-1 in MP isolated from ECs cultured with 21%O2 .

Electron microscopy and expression of endothelial nitric oxide synthase and caveolin-1 in endothelial microparticles Isolated MPs were also evaluated by TEM. A representative image of endothelial MPs by TEM is presented in Figure 1C, showing that

isolated MPs are intact and spherical vesicular structure with the size in a range of 0.1–1.0 μm in diameter as described previously [1, 16]. Since caveolae and MP were both membrane structures and eNOS is also located at caveolae, we examined protein expression for caveolae specific marker caveolin-1 and eNOS in isolated MPs. Our

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results showed that eNOS and caveolin-1 were detected in isolated endothelial MPs. Endothelial MP expression of eNOS and caveolin-1 is shown in Figure 1D.

Effects of 1,25(OH)2 D3 on caveolae protein expression in endothelial cells cultured under lowered oxygen condition Oxidative stress-induced endothelial dysfunction is considered a key event in the development and progression of cardiovascular diseases. Caveolin-1 is a major membrane protein of caveolae, a vital plasma membrane sensor that responds to plasma membrane and extracellular environment stresses. Endothelial NOS is a central regulator of cellular function that is important to maintain endothelial homeostasis, which is also expressed in caveolae. Since lipid rafts and/or caveolae play a pivotal role in MP formation [16], we then examined effects of 1,25(OH)2 D3 on caveolin-1 and eNOS expression in ECs to determine if altered caveolin-1 and eNOS expression were related to increased MP release under lowered oxygen condition. Vitamin D receptor expression was also determined. Results are shown in Figure 2A. We found that caveolin-1 expression was upregulated in cells cultured under lowered oxygen condition, P < 0.05. In contrast, eNOS expression was significantly reduced in cells cultured under lowered oxygen condition compared to cells cultured under standard culture condition, P < 0.05. This phenomenon of upregulation of caveolin-1 and downregulation of eNOS was attenuated when 1,25(OH)2 D3 was present in culture, P < 0.05. Expression of p-eNOSSer1177 and p-eNOSThr495 were also determined. Our results showed that p-eNOSSer1177 expression was reduced in cells cultured under lowered oxygen condition, P < 0.05, but p-eNOSSer1177 expression was upregulated in cells treated with 1,25(OH)2 D3 under both standard and lowered oxygen condition, P < 0.05. In contrast, p-eNOSThr495 expression was increased in cells cultured under lowered oxygen condition, P < 0.05, which could be attenuated when 1,25(OH)2 D3 was present in culture (Figure 2A). Similar to eNOS, VDR expression was downregulated in cells cultured under lowered oxygen condition, P < 0.05, but dramatically upregulated in cells treated with 1,25(OH)2 D3 in both standard and lowered oxygen culture conditions, P < 0.01. To further determine effects of 1,25(OH)2 D3 -mediated downregulation of caveolin-1 and upregulation of eNOS expression in cells challenged under lowered oxygen condition, cells were treated with different concentrations of 1,25(OH)2 D3 at 0, 10, 50, and 100 nM for 48 h. Expression of caveolin-1, eNOS, p-eNOSSer1177 , p-eNOSThr495 , and VDR was determined. Interestingly, we found that caveolin-1 expression was dose-dependently decreased and eNOS expression was dose-dependently increased in cells treated with 1,25(OH)2 D3 (Figure 2B). Expression for p-eNOSSer1177 and p-eNOSThr495 was opposite. 1,25(OH)2 D3 induced a dose-dependent increase in peNOSSer1177 expression, P < 0.01, but a dose-dependent decrease in p-eNOSThr495 expression, P < 0.05, in cells cultured under lowered oxygen condition (Figure 2B). Bar graphs are relative expression for caveolin-1, eNOS, p-eNOSSer1177 , p-eNOSThr495 , and VDR expression after normalization by β-actin. These findings suggest that vitamin D could modulate eNOS and caveolin-1 expression in ECs, especially under oxidative stress challenge.

Effects of 1,25(OH)2 D3 on Akt and ERK expression in endothelial cells cultured under lowered oxygen condition To study the potential mechanism of 1,25(OH)2 D3 mediated upregulation of eNOS expression, we determined Akt pathway molecules

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Akt and p-AktSer473 and MAPK/ERK pathway molecules ERK and p-ERK expression in ECs cultured under standard or lowered oxygen conditions with or without 1,25(OH)2 D3 treatment. Results are shown in Figure 3A. We found that p-AktSer473 expression was significantly reduced in cells cultured under lowered oxygen condition, P < 0.05, and p-AktSer473 expression was upregulated in cells treated with 1,25(OH)2 D3 in both standard and lowered oxygen culture conditions, P < 0.05. Phospho-ERK expression was also significantly reduced, P < 0.01, but not affected by 1,25(OH)2 D3 , in cells cultured under lowered oxygen condition. There was no difference for Akt and ERK expression in cells cultured under standard and lowered oxygen condition with or without 1,25(OH)2 D3 treatment. We also assessed the dose–response effects of 1,25(OH)2 D3 on Akt, p-AktSer473 , ERK, and p-ERK expression in cells cultured under lowered oxygen condition. We found that total Akt expression was not changed, but p-AktSer473 expression was dose-dependently increased, in cells treated with 1,25(OH)2 D3 (Figure 3B), P < 0.05 and P < 0.01, respectively. ERK and p-ERK expression was not affected (Figure 3B). The bar graphs represent relative Akt, p-AktSer473 , ERK, and p-ERK expression. These results suggest that 1,25(OH)2 D3 could promote Akt phosphorylation, but had no effect on ERK expression in ECs.

1,25(OH)2 D3 inhibits ROCK cleavage in endothelial cells under oxidative challenge ROCK1 is a major downstream effector of the small GTPase RhoA. It is a regulator of the actomyosin cytoskeleton and promotes contractile force generation. Truncated ROCK1 without its carboxyterminal inhibitory domain would increase ROCK1 activity [17]. Abnormal ROCK1 activation was found to play a critical role in MP formation and/or release [18]. To further explore whether vitamin D could also affect ROCK1, we examined ROCK1 expression in cells cultured under standard and lowered oxygen culture conditions. Results are shown in Figure 4A. For ROCK1 expression, two bands were detected: one at 160 kD and one at 130 kD. The 160 kD is considered as its native form and the 130 kD one is considered as cleaved ROCK1, which loses its carboxy-terminal domain after it is activated [19]. We found that although native form of ROCK1 was not significantly affected in cells cultured under lowered and standard oxygen condition with or without 1,25(OH)2 D3 in culture, cleaved ROCK1 expression was increased when cells were cultured under lowered oxygen condition, P < 0.05, and this lowered oxygen induced increased ROCK1 cleavage could be attenuated when cells were treated with 1,25(OH)2 D3 , P < 0.05. The inhibitory effect of 1,25(OH)2 D3 on ROCK1 cleavage was further confirmed in the dose-dependent study, in which cells were cultured under lowered oxygen condition (Figure 4B). These results indicated that vitamin D could inhibit ROCK1 activation, which plays, at least in part, a role in controlling of MP formation and/or release in ECs under oxidative stress stimulation.

Effect of 1,25(OH)2 D3 on endothelial nitric oxide synthase and caveolin-1 expression in endothelial microparticles As shown in Figure 1D, expression of eNOS and caveolin-1 was detected in isolated MPs. Since MPs are considered as the “shuttle” that carry components of plasma membrane of parental cells and exert biological function in regulation of cellular function in the cardiovascular system [20], we then determined if endothelial MP eNOS and caveolin-1 expression could be affected in cells treated with 1,25(OH)2 D3 . Figure 5 shows eNOS and caveolin-1 protein

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Figure 2. Protein expression for caveolin-1, eNOS, p-eNOSSer1177 , p-eNOSThr495 , and VDR in ECs cultured under 21%O2 or 2%O2 in the presence or absence of 1,25(OH)2 D3 . A) Representative western blots for caveolin-1, eNOS, p-eNOSSer1177 , p-eNOSThr495 , and VDR expression. β-Actin expression was determined and showed equal loading of total cellular protein for each sample. The bar graphs show relative protein expression after normalization with β-actin expression. Caveolin-1 expression was increased, but eNOS expression was reduced, in cells cultured under 2%O2 vs. 21%O2 , ∗ P < 0.05. The lowered oxygen induced downregulation of eNOS and upregulation of caveolin-1 could be inhibited by 1,25(OH)2 D3 , # P < 0.05. p-eNOSSer1177 expression was reduced, but p-eNOSThr495 expression was increased, in cells cultured under 2%O2 vs. 21%O2 , ∗ P < 0.05. 1,25(OH)2 D3 could upregulate p-eNOSSer1177 expression in cells treated with 1,25(OH)2 D3 in both 21%O2 (∗ P < 0.05) and 2%O2 (# P < 0.05) conditions. 1,25(OH)2 D3 could upregulate VDR in both conditions when compared with the cells cultured under the same condition without 1,25(OH)2 D3 , ∗ ∗ P < 0.01 and # # P < 0.01, respectively. B) Western blots of endothelial caveolin-1, eNOS, p-eNOSSer1177 , p-eNOSThr495 , and VDR protein expression in cells cultured under 2%O2 in the presence of different concentrations of 1,25(OH)2 D3 . The bar graphs show relative protein expression after normalization with β-actin expression. 1,25(OH)2 D3 induced a dose-dependent decrease in caveolin-1 expression, # P < 0.05 and ## P < 0.01, but a dose-dependent increase in eNOS expression, respectively. p-eNOSSer1177 expression was dose-dependently upregulated, but p-eNOSThr495 expression was dose-dependently downregulated in cells treated with 1,25(OH)2 D3 . Vitamin D receptor expression was also upregulated, # # P < 0.01. Data are means ± SE from five independent experiments.

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Figure 3. Protein expression for Akt, p-AktSer473 , ERK, and p-ERK in ECs cultured under 2%O2 or 21%O2 in the presence or absence of 1,25(OH)2 D3 . A) Representative blots for Akt, p-AktSer473 , ERK, and p-ERK expression. β-Actin expression was also determined to show equal loading of total cellular protein for each sample. The bar graphs show relative protein expression for Akt, p-AktSer473 , ERK, and p-ERK expression after normalization with β-actin expression. p-AktSer473 and p-ERK expression were significantly downregulated in cells cultured under 2%O2 vs. 21%O2 , ∗ P < 0.05 and ∗ ∗ P < 0.01, respectively. p-AktSer473 expression was upregulated in cells treated with 1,25(OH)2 D3 in both 21%O2 and 2%O2 culture conditions. In contrast, 1,25(OH)2 D3 had no effect on p-ERK expression. Both Akt and ERK expression were not affected in cells cultured under 21%O2 vs. 2%O2 with or without 1,25(OH)2 D3 treatment. B) Akt, p-AktSer473 , ERK, and p-ERK expression in cells cultured under 2%O2 treated with different concentrations of 1,25(OH)2 D3 . The bar graphs show relative protein expression after normalization with β-actin expression. p-AktSer473 expression was dose-dependently upregulated by 1,25(OH)2 D3 vs. untreated control cells, # P < 0.05, and ## P < 0.01. Data are means ± SE from five to six independent experiments.

expression in MPs isolated from ECs cultured under standard or lowered oxygen condition in the presence or absence of 1,25(OH)2 D3 . Microparticle eNOS expression was reduced from cells cultured under lowered oxygen condition, but reduced eNOS expression was somewhat attenuated when cells were treated with 1,25(OH)2 D3 . Microparticle caveolin-1 expression was not much changed from cells cultured under standard or lowered oxygen conditions with or without treatment of 1,25(OH)2 D3 . Because of the lack of housekeeping protein in MPs such as β-actin in cellular protein and the fact of direct relationship between eNOS and caveolin-1 [21], the ratio of eNOS and caveolin-1 expression was then determined and results are shown in the bar graph of Figure 5. The ratio of MP eNOS and caveolin-1 expression was significantly reduced in cells cultured under lowered oxygen condition compared to cells incubated under

standard culture condition, P < 0.01, but partially reversed in cells treated with 1,25(OH)2 D3, P < 0.05, respectively. These data suggest that in addition to amount of MPs, 1,25(OH)2 D3 could also modulate the “composition” of MP released by ECs.

Discussion In this study, using oxidative stress-induced MP release as the testing model, we investigated protective effects of vitamin D on ECs. We specifically determined if 1,25(OH)2 D3 could suppress MP release mediated by oxidative stress in cells cultured under lowered oxygen condition. Using annexin-V as the marker of released MPs assessed by flow cytometry, we found that ECs released significantly more MPs when cells were incubated under lowered oxygen condition

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Figure 4. Protein expression for ROCK1 and truncated ROCK1 in ECs cultured under 2%O2 or 21%O2 in the presence or absence of 1,25(OH)2 D3 . A) Western blot for ROCK1 and cleaved ROCK1 expression. β-Actin expression was determined to show equal loading of total cellular protein for each sample. The bar graphs show relative protein expression for ROCK1 and cleaved ROCK1 after normalization with β-actin expression. ROCK1 had no significant difference among the groups, the cleaved ROCK1 was increased in cells cultured under 2%O2 , ∗ P < 0.05, whereas cleaved ROCK1 expression was reduced when 1,25(OH)2 D3 was present in cultured, # P < 0.05. B) Western blots of native and cleaved ROCK1 expression in ECs cultured under 2%O2 treated with different concentrations of 1,25(OH)2 D3 . The bar graphs show relative protein expression after normalization with β-actin expression. 1,25(OH)2 D3 had no significant effect on native ROCK1 expression, but could reduce cleaved ROCK1 expression in a dose-dependent manner, # # P < 0.01. Data are means ± SE from five independent experiments.

and increased MP release was accompanied by increased superoxide generation. We further found that 1,25(OH)2 D3 could not only suppress MP release but also inhibit superoxide generation when cells were cultured under lowered oxygen condition. Although endothelial MPs are involved in cellular communication under physiological condition [1], increased MP release by dysfunctioned ECs has been demonstrated to play a crucial role in the development and pathogenesis in many cardiovascular diseases such as hypertension, atherosclerosis, ischemic myocardial syndromes, and diabetes [22, 23]. Increased MP shedding by ECs has significant impact on vascular homeostasis. They promote platelet aggregates, impair angiogenesis, and affect vascular tone by impairing Ach-induced vasorelaxation and nitric oxide production, and they could also produce superoxide radicals [24, 25]. Therefore, our finding of vitamin D

suppression of endothelial MP release induced by oxidative stress provides a conceivable evidence of vitamin D in the protection of vascular ECs. Furthermore, since oxidative stress is well known as one of the most potent inducers of endothelial dysfunction, the finding of the inhibitory effect on superoxide generation by 1,25(OH)2 D3 also suggests vitamin D antioxidative property in the cardiovascular system. Although we did not test specific antioxidants on MP release in our study, suppression of MP release by antioxidants was reported previously in HUVECs and in retinal pigment epithelial cells [26, 27]. Many investigators have used 1 or 2%O2 , 5% CO2 and balance with N2 as lowered oxygen or hypoxic condition for their oxidative stress or lowered oxygen condition studies, although this condition may not be exactly the oxygen tension in in vivo organs. A study

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Figure 5. Effect of 1,25(OH)2 D3 on eNOS and caveolin-1 expression in MPs isolated from ECs. A) Representative western blot of eNOS and caveolin-1 expression in MPs isolated from ECs cultured under 2%O2 and 21%O2 in the presence or absence of 1,25(OH)2 D3 . B) Relative ratio of eNOS and caveolin-1 expression in endothelial MPs. The ratio of eNOS and caveolin-1 expression was dramatically decreased in MPs released from cells cultured under 2%O2 , ∗∗ P < 0.01. 1,25(OH)2 D3 had no significant effect on the ratio of MP eNOS and caveolin-1 expression in cells cultured under 21%O2 , but could increase the ratio of MP eNOS and caveolin-1 expression in cells cultured under 2%O2 , # P < 0.05. Data are means ± SE from five independent experiments.

conducted by Mackova et al. provided valuable information [28]. These investigators measured oxygen tension in varied oxygenbuffered medium. They found that oxygen tension was 14–16 mmHg in a 2%O2 incubator, 34–42 mmHg in a 5%O2 incubator, and 138–142 mmHg in an air incubator (air/5%CO2 = 21%O2 ). They concluded that these measurements approximate the Henry Law theoretical value of 15, 38, and 140 mmHg. Their data suggest that an approximate 2%O2 could be an optimal hypoxic condition and 21%O2 environment may be a favorable condition to study cell function in vitro. A study conducted by Kay et al. also provided valuable information [29]. They studied glucose metabolism and hormone release by placental villous tissue in 0%, 20%, and 95%O2 conditions in an in vitro perfusing system and found that glucose consumption was lowest in tissues with 0%O2 , and both lactate and lactate dehydrogenase release were lowest in tissues with 95%O2 , while tissues with 20%O2 provided an optimal support for hormonal release and functional performance. Their data suggest that an approximate 20%O2 environment may be a favorable condition to study trophoblast function in vitro. We also published several studies using 2%O2 as a hypoxic condition to study placental functions [30–32]. Therefore, we believe that using 2%O2 as a hypoxic condition to study oxidative stress-mediated endothelial function is appropriate. HUVECs were used in this study. Many published works showed that there were phenotypic and functional differences between HUVECs and ECs derived from systemic vasculature [33–36]. Although

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HUVECs and ECs from different organs or vasculature (artery or vein) might respond differently to hypoxic stimulation [33, 37], the evaluation of hypoxic response between “maternal” endothelium and HUVECs is lacking. However, studies published by our group and the others did show that some characteristics of HUVECs from preeclamptic pregnancies such as increased inflammatory responses and barrier dysfunction are in lines with what were reported in the systemic vasculature in preeclamptic as compared to normal pregnancies [13, 38–41]. It is expected that HUVECs from preeclamptic pregnancies may release more MPs than cells from normal pregnancies, which warrants further investigation. Microparticle forms when cells lost asymmetrical distribution of membrane lipids between the inner and outer leaflets of a plasma membrane and shed MPs are composed of a phospholipid bilayer containing transmembrane proteins, receptors, enzymes, and mRNA derived from their parental cells [42]. Caveolae is an important lipid raft and functions as the center of signal transduction and endocytosis, which is enriched in ECs. Burger et al. found depletion of membrane lipid rafts and/or caveolae attenuated endothelial MP generation induced by angiotensin II [16], revealing the important role of cholesterol-rich membrane microdomains in MP formation. To explore whether isolated MPs possess endothelial membrane element caveolae, we determined caveolin-1 and eNOS expression in isolated MPs. Caveolin-1 is the major scaffolding protein and a specific marker of caveolae. Endothelial NOS is enriched in caveolae. Our results revealed that abundant caveolin-1 and eNOS were detected in isolated MPs and the ratio of eNOS and caveolin-1 protein expression in MPs was reduced in cells cultured under lowered oxygen condition. We further noticed that reduced ratio of eNOS and caveolin-1 in endothelial MPs could be partially inhibited by 1,25(OH)2 D3 . Although the reason for the proportional change in eNOS and caveolin-1 in endothelial MPs is not known, our data indicated that altered eNOS and caveolin-1 expression in endothelial MPs is a consequence of endothelial oxidative stress. This notion is in line with the findings that were reported by Horn et al. [43]. By measuring eNOS-dependent NO production in isolated MPs, these investigators demonstrated that circulating MPs carry functional eNOS. They also found that endothelial MPs from patients with cardiovascular diseases expressed less eNOS than those from healthy controls [43], suggesting that “composition” changes in MPs is a consequence of endothelial dysfunction in cardiovascular diseases. Based on these observations, we hypothesized that increased MP release by cells cultured under lowered oxygen condition is pertinent to altered eNOS and caveolin-1 expression in ECs and we tested if vitamin D could preserve eNOS expression and protect endothelial homeostasis. As expected, we found that eNOS expression was significantly reduced and caveolin-1 expression was significantly increased in cells cultured under lowered oxygen condition and this oxidative stress induced altered eNOS and caveolin-1 expression could be prevented by 1,25(OH)2 D3 treatment. In addition, similar to eNOS expression, VDR expression was also downregulated in cells cultured under lowered oxygen condition and upregulated when 1,25(OH)2 D3 was present in culture. It is well known that most of the biological actions of vitamin D are initiated by activation of its receptor VDR. Although we did not directly study the interaction of VDR and eNOS in this study, upregulation of eNOS expression induced by 1,25(OH)2 D3 could be due to the presence of VDR element in eNOS promoter. This was demonstrated previously by Mart´ınez-Miguel et al. in EA.hy926 ECs [44]. In addition, a relationship of eNOS and caveolin-1 was also reported. Using yeast two-hybrid system, Ju et al. demonstrated a direct

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interaction between caveolin-1 and eNOS in ECs [21]. They found that the interaction of eNOS with caveolin-1 fusion proteins significantly inhibited eNOS catalytic activity [21]. Thus, inhibition of caveolin-1 expression may lead to less eNOS and caveolin-1 association and as a result, promote eNOS function in ECs. A study conducted by Fleming et al. revealed that bradykinin could enhance the phosphorylation of eNOS at Ser1177, but dephosphorylation of eNOS at Thr495 in HUVECs [45], which suggests that phosphorylation of eNOS at Thr495 has an opposite effect on eNOS activity as to phosphorylation of eNOS at Ser1177 in ECs [45]. In this study, a similar observation was found in cells cultured under lowered oxygen condition treated with 1,25(OH)2 D3 , i.e., 1,25(OH)2 D3 promoted p-eNOSSer1177 expression and attenuated p-eNOSThr495 expression. It is known that eNOS activity is largely regulated by phosphorylation at Ser1177 [46]. A recent study also found that NO could limit endothelial MP release by remodeling of plasma membrane and cytoskeletal reorganization [47]. Therefore, 1,25(OH)2 D3 induced upregulation of p-eNOSSer1177 expression and repression of p-eNOSThr495 expression could be pivotal in the regulation of NO generation and suppression of MP formation and/or shedding by ECs. It is known that the phosphorylated relative to the total pool of a protein is the true indicator of activation. Thus, we also calculated the eNOS activity as (p-eNOSSer1177 /eNOS)/(peNOSThr495 /eNOS). The result revealed that eNOS activity was reduced in cells cultured under lowered oxygen condition but increased in cells treated with 1,25(OH)2 D3 in both standard and lowered oxygen culture conditions (data not shown), which was consistent to the relative protein expression for eNOS and p-eNOSSer1177 that were normalized with β-actin expression as shown in Figure 2. It was reported that phosphorylation of eNOS at Ser1177 could be induced by p-AktSer473 [46]. To further explore the regulatory mechanism of 1,25(OH)2 D3 on eNOS, expression of Akt and pAktSer473 was then determined. We found that similar to eNOS and p-eNOSSer1177 , 1,25(OH)2 D3 could inhibit oxidative stress induced downregulation of p-AktSer473 expression in cells cultured under lowered oxygen condition. Moreover, we found that 1,25(OH)2 D3 also induced a dose-dependent increases in eNOS as well as p-eNOSSer1177 and p-AktSer473 expression in cells cultured under lowered oxygen condition. However, 1,25(OH)2 D3 had no effect on ERK and pERK expression in cells cultured under lowered oxygen condition, suggesting that 1,25(OH)2 D3 may not regulate ERK signaling pathway molecules. Similar to eNOS activity, the changes in AKT and ERK activities (p-AktSer473 /AKT and pERK/ERK) (data not shown) were also consistent to their relative protein expression as normalized with β-actin for p-AktSer473 and pERK as shown in Figure 3. Since pAktSer473 is considered an activator of p-eNOSSer1177 , 1,25(OH)2 D3 induced increase in p-AktSer473 expression could be, at least in part, a mechanism of vitamin D induced upregulation of p-eNOSSer1177 expression and upregulation of p-AktSer473 and p-eNOSSer1177 expression is likely an important signaling machinery of vitamin D protective actions on vascular ECs. ROCK1, which mediates downstream of RhoA, is a key regulator of actin–myosin contraction and involved in a wide range of cellular functions such as adhesion, migration, and proliferation [48]. Abnormal activation of ROCK1 was found to be associated with endothelial dysfunction and contributes to the development of many cardiovascular diseases [49]. Studies also showed that there is a striking crosstalk between ROCK and eNOS signaling in regulation of endothelial function [50, 51]. RhoA/ROCK could not only downregulate eNOS expression by altering eNOS mRNA stability

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and half-life, but also inhibit eNOS activity by inactivation of Akt and de-phosphorylation of eNOS at Ser-1177 [50, 51]. In contrast, both in vitro and in vivo studies found that exogenous NO had ability to inhibit ROCK activity in vascular smooth muscle cells [52]. In this study, we examined ROCK1 expression and found that 1,25(OH)2 D3 could inhibit ROCK cleavage induced by oxidative stress. As mentioned earlier, activation of ROCK1 induces assemble of actin–myosin network, which is directly associated with MP release [53, 54]. Thus, the inhibitory effect on ROCK1 activation and/or cleavage by 1,25(OH)2 D3 may also be a part of the mechanisms of vitamin D in suppression of MP formation and/or release in ECs. In summary, we have demonstrated, for the first time, that 1,25(OH)2 D3 could suppress MP release and superoxide generation induced by oxidative stress in ECs. The protective effects of 1,25(OH)2 D3 on ECs could be via the following mechanisms: (1) modulation of eNOS and caveolin-1 expression in MPs and ECs, (2) increase in endothelial eNOS expression and/or activity by phosphorylation of eNOSSer1177 through upregulation of p-AktSer473 signaling, and (3) inhibition of ROCK1 cleavage to stabilize cytoskeletal reorganization. It is known that endothelial MP shedding is increased in cardiovascular diseases [4]. Maternal MP levels are also increased in pregnant women complicated with preeclampsia and the increased endothelial MPs were found to be associated with severity of this pregnancy disorder [6]. Although a direct relationship between maternal vitamin D and MP levels in normal pregnancy and preeclampsia is lacking, epidemiology studies have shown that sufficient maternal vitamin D levels are associated with reduced pregnancy disorders such as preeclampsia and preterm delivery [55, 56]. Moreover, animal studies also demonstrated regulation of vascular eNOS activity and inhibition of NFκB and RhoA/Rho kinase by vitamin D [57, 58]. Thus, results obtained from this study would provide new insights into the mechanism of vitamin D in the protection of ECs against oxidative stress-induced cell injury in cardiovascular diseases and pregnancy disorders. Disclosure: None

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Vitamin D suppresses oxidative stress-induced microparticle release by human umbilical vein endothelial cells.

Endothelial microparticle (MP) release was increased in numerous cardiovascular diseases including preeclampsia. Oxidative stress is a potent inducer ...
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