INTRODUCTION

Cardiovascular (CV) diseases remain the leading cause of death in modern societies, with endothelial dysfunction being the common pathology of CV diseases with various etiologies. As important nontraditional risk factors for CV events, low levels of vitamin D and its analogs have been associated with endothelial dysfunction in diverse groups of people, including healthy individuals[1,2] and patients with chronic kidney disease (CKD),[3-6] diabetes,[7] lupus,[8] stroke,[9] obesity,[10] and rheumatoid arthritis.[11] Although the physiological mechanisms linking vitamin D deficiency to endothelial dysfunction in these contexts have not been established, they may involve vascular inflammation. During the inflammatory process, endothelial cells become activated.[12] Endothelial activation induces the upregulation of adhesion molecules, such as E-selectin and vascular cell adhesion molecule-1 (VCAM-1), which play pivotal roles in leukocyte-endothelium interactions, eventually leading to atherosclerosis and CV diseases.

Tumor necrosis factor-α (TNF-α) is one of the primary mediators of endothelial activation, which contributes to the inflammatory endothelial cell response and is initiated through activation of the classical nuclear factor kappa-B (NF-κB) pathway.[13] Vitamin D has been shown to have anti-inflammatory effects on various diseases. In healthy women, the serum 25(OH)D concentration is negatively correlated with the TNF-α concentration.[14] Supplementation with paricalcitol, a vitamin D analog, was associated with a reduction in the serum levels of TNF-α in CKD patients.[15] The relationship between vitamin D and NF-κB signaling has also been reported. Suzuki et al. reported that in human coronary arterial endothelial cells (HCAECs), vitamin D inhibits the activation of the NF-κB signaling pathway as well as the expression of its downstream target E-selectin.[16] However, it is still unknown which stage of the NF-κB pathway is affected by vitamin D in endothelial cells.

To further clarify the effects of 1α, 25-dihydroxyvitamin D3 (1α, 25(OH)2D3) on the development of CV diseases and the underlying mechanisms, we conducted an in vitro study to evaluate the effects of 1α, 25(OH)2D3, the active form of vitamin D3, on TNF-α-induced adhesion molecule expression in human endothelial cells. We also explored the effects of 1α, 25(OH)2D3 on various stages of the NF-κB pathway, including early activation and VCAM-1 and E-selectin promoter binding, to provide additional insight into the molecular mechanisms linking vitamin D to endothelial function.

MATERIAL AND METHODS Cell culture

Normal cryopreserved human umbilical vein endothelial cells (HUVECs) (KGG3102-1, KeyGEN BioTECH, Nanjing, China) were rapidly thawed in a 37–40°C water bath and grown in F12K medium (KGL1903-500, KeyGEN BioTECH, Nanjing, China) at 37°C in a 5% carbon dioxide humid incubator. When the cells reached 80% confluence, they were passaged with 0.25% trypsin (KGA1519-5, KeyGEN BioTECH, Nanjing, China). Cells in passages 3–4 were used for experiments. Mycoplasma and short tandem repeat tests were performed on the cells used in this study.

Cytokine treatment

HUVECs were incubated with various concentrations of 1α,25-(OH)2D3 (740578; Sigma‒Aldrich, USA) for 30 min and then exposed to 40 ng/mL TNF-α (300-01A; PeproTech Inc., USA) for 24 h unless otherwise indicated.[17] To block NF-κB signaling, HUVECs were pretreated with the specific NF-κB inhibitor NF-κB SN50 (213546-53-3, MCE, USA) for 1 h and then incubated with 40 ng/mL TNF-α for 24 h.[18] To assess NF-κB early activation, HUVECs were pretreated with 1α,25-(OH)2D3 for 30 min, followed by incubation with TNF-α for various periods as indicated.

Transfection with vitamin D receptor-siRNA (VDR-siRNA)

HUVECs were seeded into 24-well plates (3422, Corning Incorporated, USA) at a density of 5.0 × 105 cells per well in 500 μL of antibiotic-free medium and incubated for 24 h before transfection. The cells were then incubated with a mixture of 2 siRNAs (200 nM final concentration), medium, and Lipofectamine 2000 buffer (11668027, Invitrogen, Carlsbad, CA, USA) for 4 h; washed; and incubated for an additional 48 h at 37°C. The nucleotide sequences of the 2 VDR-siRNAs are 5’-UCCGUGCCUCTGGCTTTCUCTTCUUTT-3’ and 5’-AGCGCATCATTGCCATACT-3’, targeting sequences at loci 842 and 793 of the human vitamin D receptor (VDR) gene, respectively.

Ribonucleic acid (RNA) isolation and quantitative real-time polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from HUVECs using TRIzol reagent (15596018CN; Invitrogen, USA) according to the manufacturer’s instructions. Total RNA was reverse transcribed to complementary deoxyribonucleic acid (cDNA) using the SuperScript IV CellsDirect cDNA Synthesis Kit (11750150; Invitrogen, USA). cDNAs synthesized from total RNA were used as templates for polymerase chain reaction (PCR) amplification with gene-specific primer pairs. The sequences of the primer sets were as follows: E-selectin, 5’-CCGAGCGAGGCTACATGAAT-3’ (forward) and 5’-GAGAACTCACAGCTGGACCC-3’ (reverse); VCAM-1, 5’-TCGTGATCCTTGGAGCCTCA-3’ (forward) and 5’-AGGAAAAGAGCCTGTGGTGC-3’ (reverse); VDR, 5’-GCTTGTCAAAAGGCGGCAG-3’ (forward) and 5’-CCCAAAGGCTTCTGGTCCG-3’ (reverse); and β-actin, 5’-TCACCATGGATGATGATATCGC-3’ (forward) and 5’-ATAGGAATCCTTCTGACCCATGC-3’ (reverse). The internal control for the PCR was β-actin. PCR was performed in an Applied Biosystems Step One Plus RT-PCR system (v1.0, Invitrogen, USA). The 2−ΔΔCT method was used to calculate the relative expression of E-selectin, VCAM-1, and the VDR.

Western blot analysis

The preparation of whole-cell lysates and Western blot analysis of protein expression were carried out using routine procedures. Briefly, after various treatments as indicated, HUVECs were washed with ice-cold phosphate buffer saline (PBS) and lysed in the presence of protease inhibitor cocktail (P8215-1ML; Sigma‒Aldrich, USA) for 15 min in ice-cold lysis buffer (KGB5202-100, KeyGEN BioTECH, Nanjing, China). The cell extracts were centrifuged at ×14,000 g for 15 min in a precooled centrifuge at 4°C, and the supernatants were stored at −70°C. For the detection of proteins in the nucleus, the nuclei were first isolated using the Nuclei Extraction Kit (Nuclei PURE Prep, NUC201-1KT, Merck) and then lysed as described above. Protein was quantified through a bicinchoninic acid (BCA), A65453, Invitrogen, USA) kit: an appropriate amount of sodium dodecyl sulfate (SDS) was added to a final concentration of 0.1% and then diluted to different concentrations of the standard protein solution. Different concentrations of standard protein mixture were added to a 96-well plate, and each solution with different concentrations was pipetted into 3 wells. BCA working solution was added to each well and mixed well. The optical density (OD) value (450 nm) was measured, and the standard curve was plotted. For Western blot analysis, protein lysates were separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels (NP0346BOX, Sigma‒ Aldrich, USA) and transferred to nitrocellulose membranes. Primary antibodies were added, the proteins were blocked overnight (4°C), and secondary antibodies were added the next day, and the proteins were washed 3 times with PBS. Proteins were visualized through horseradish peroxidase detection reagents according to the manufacturer’s instructions (74924, New-SUPER ECL, KeyGEN BioTECH, Nanjing, China) and exposed to autoradiographic film (G: BOX chemiXR5). Gel-Pro32 software (v4.0, Media Cybernetics, USA) was used to grayscale the results. The primary antibodies anti-VCAM-1 (1:1000, ab134047), anti-E-selectin (1:1000, ab18981), anti-p65 NF-κB (1:1000, ab32536), anti–phospho-p65 NF-κB (phospho-S529) (1:1000, ab109458), anti-IκBα (1:1000, ab32518), anti– phospho-IκBα (1:1000, phospho-S36) (ab133462), anti-VDR (1:1000, ab109234), and H3 (1:1000, ab1791) were obtained from Abcam (Cambridge, UK).

Chromatin immunoprecipitation (ChIP) assays

NF-kB binding to the VCAM-1 and E-selectin gene promoters in HUVECs was determined by ChIP assays using a commercially available ChIP assay kit (P2078, Lake Placid, NY, USA). Briefly, HUVECs subjected to various treatments as indicated were treated with 1% formaldehyde to cross-link histones to DNA and then treated with a stop buffer for 5 minutes to stop cross-linking. The chromatin was extracted and fragmented by sonication. The sonicated chromatin was incubated with an anti-p65 antibody (1:1000, ab218533, Abcam, UK) overnight at 4°C, followed by incubation with protein A–agarose for 2 h. The precipitates were washed, and the chromatin complexes were eluted. After reversal of the cross-linking, the DNA was purified and used as a template for PCR with primers flanking the NF-kB binding sites in the VCAM-1 and E-selectin gene promoters. The sequences of the primers used for the ChIP assay were as follows: VCAM-1, 5’- GAGGAGCAGGTAGGACTT-3’ (forward), and 5’- CTGAGGTCTGGAATCTATAACT-3’ (reverse) and E-selectin, 5’-GCCTCTCACCTCAGCCTTGTAG-3’ (forward), and 5’-ACATTGTGCCAACATCAGTATCCT-3’ (reverse). The PCR products were run on a 1.5% agarose gel and stained with ethidium bromide.

Coimmunoprecipitation

Coimmunoprecipitation was carried out using an established method. Briefly, HUVECs were transfected with the NF-κB expression vector using Lipofectamine 2000 reagent (11668027; Invitrogen, Carlsbad, CA, USA) for 48 h and then incubated with or without 40 ng/mL TNF-α and/or 1α,25-(OH)2D3 (10−8) for 1 h before being subjected to coimmunoprecipitation. The cells were lysed in 1 mL of non-denaturing lysis buffer containing 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor, and 5 μL of 100 mM PMSF. The cell lysates were incubated with 2 μg of an anti-VDR antibody overnight at 4°C, followed by precipitation with 20 μL of Protein G agarose for 3 h at 4°C. The precipitated complexes were separated on 10% SDS‒PAGE gels and immunoblotted with an anti-p65 NF‒κB antibody.

Immunofluorescence staining

HUVECs were pretreated with or without 10−8 M 1,1α, 25(OH)2D3 for 24 h and then exposed to 40 ng/mL TNF-α for 1 h.[19] The cells were fixed with 4% paraformaldehyde for 30 min and incubated with normal goat serum for 20 min to block non-specific binding of the antibodies. Afterward, the cells were stained with an anti-p65 antibody for 2 h in a humidified chamber, followed by staining with fluorescein isothiocyanate-conjugated secondary antibody (ab6717, Abcam, Cambridge, UK) for 1 h in the dark. Each slide was stained with 4,6-diamidino-2-phenylindole (DAPI) (ab104139, Abcam, Cambridge, UK) for 5 min to visualize the nucleus. The slides were viewed with an Olympus IX51 microscope equipped with a digital camera (Japan). The fluorescence intensity of NF-κB p65 was quantified using ImageJ software (v1.8.0, National Institutes of Health, USA). In addition, after isolation of the nuclei and cytoplasm, p65 NF-κB expression was detected using Western blotting as described above.

Statistical analyses

Analyses were performed by the Statistical Package for the Social Sciences statistics (v22, IBM, USA). The results are presented as the mean ± standard deviation. Comparisons between two groups were made using independent samples t-tests, and comparisons between multiple groups were made using analysis of variance for repeated measures and the least significant difference within-group tests. Statistical significance was assumed at a two-tailed value of P < 0.05.

RESULTS Treatment of HUVECs with 1α,25-(OH)2D3 inhibits TNF-α-induced VCAM-1 and E-selectin release

First, we examined the expression of the VDR in cells after transfection with VDR-siRNA by PCR and Western blotting. As shown in Figure 1a, we observed that the expression of the VDR in HUVECs was significantly reduced after both VDR-siRNA intervention treatments (P < 0.05), confirming the successful knockdown of VDR expression by VDRsiRNA. When HUVECs were stimulated with 40 ng/mL TNF-α for 24 h, the expression levels of VCAM-1 and E-selectin were significantly increased. Pre-treatment of HUVECs with various concentrations of 1α,25-(OH)2D3 for 30 min markedly reduced VCAM-1 and E-selectin mRNA and protein levels, with the maximum reduction observed at moderate concentrations (10−8 M 1α, 25-(OH)2D3) [Figure 1b-d]. In HUVECs transfected with a specific VDRsiRNA, the inhibitory effects of 1α,25-(OH)2D3 on VCAM-1 and E-selectin expression were abrogated. The expression of VCAM-1 and E-selectin was also significantly increased in HUVECs pretreated with 40 ng/mL TNF-α and significantly inhibited in HUVECs pretreated with 10−8 M 1α or 25-(OH)2D3. Therefore, vitamin D can suppress the induction of adhesion molecule expression by TNF-α in HUVECs. After cells were transfected with a specific VDR-siRNA, the inhibitory effects of 1α,25-(OH)2D3 on the expression of adhesion molecules were abrogated [Figure 1e and f], demonstrating that this inhibition is VDR dependent.

Figure 1: Treatment of HUVECs with 1α,25-(OH)2D3 inhibits TNF-α-induced VCAM-1 and E-selectin release. (a) Effect of VDR-siRNA on VDR expression; ✶ denotes P < 0.05 compared with the control. (b) Protein imprinting map. (c) Effects of 1α,25-(OH)2D3 on adhesion molecule expression in HUVECs (Western blotting). (d) Effects of 1α,25-(OH)2D3 on adhesion molecule expression in HUVECs (PCR). ✶ denotes P < 0.05 compared with cells treated with the blank, # denotes P < 0.05 compared with cells induced with TNF-α, & denotes P < 0.05 compared with cells treated with 1α,25-(OH)2D3. (e) Protein imprinting map. (f) Effects of 1α,25-(OH)2D3 on HUVEC adhesion molecule expression at different intervention times. ✶ denotes P < 0.05 compared with cells treated with the blank, # denotes P < 0.05 compared with cells induced with TNF-α, & denotes P < 0.05 compared with cells treated with 1α,25-(OH)2D3. TNF-α: Tumor necrosis factor-α, VDR-siRNA: Vitamin D receptor siRNA, 1α, 25(OH)2D3: 1α,25-dihydroxyvitamin D3, VCAM-1: Vascular cell adhesion molecule-1, HUVECs: Human umbilical vein endothelial cells, PCR: Polymerase chain reaction.

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Effect of 1α,25-(OH)2D3 on the NF-κB signaling pathway

The NF-κB signaling pathway plays an important role in the process of endothelial activation. To confirm this, HUVECs were incubated with 40 ng/mL TNF-α in the absence or presence of a specific NF-κB inhibitor (NF-κB SN50). We found that inhibition of NF-κB signaling reduced TNF-α-induced VCAM-1 and E-selectin expression in these cells, indicating that intact NF-κB signaling is required for the activation of HUVECs [Figure 2a and b]. Since IκBα is the major inhibitor of NF-κB that binds to p65 NF-kB and blocks its activation, we further explored the effects of 1α, 25(OH)2D3 on IκBα phosphorylation, as well as p65 NF-κB phosphorylation and activation. When HUVECs were stimulated with 40 ng/mL TNF-α for 24 h, IκBα, and p65 NF-κB were rapidly phosphorylated. Pre-treatment of HUVECs with various concentrations of 1α,25-(OH)2D3 for 30 min markedly inhibited IκBα and p65 NF-κB phosphorylation, with the maximum reduction observed at moderate concentrations (10−8 M 1α, 25-(OH)2D3). In HUVECs transfected with a specific VDR-siRNA, the inhibitory effects of 1α,25-(OH)2D3 on IκBα and p65 NF-κB phosphorylation were abrogated [Figure 2c]. In addition, IκBα and p65 NF-κB were rapidly phosphorylated in HUVECs pretreated with 40 ng/mL TNF-α and significantly inhibited their phosphorylation in HUVECs pre-treated with 10−8 M 1α,25-(OH)2D3. After the cells were transfected with a specific VDR-siRNA, the inhibitory effects of 1α,25-(OH)2D3 on IκBα and p65 NF-κB phosphorylation were abrogated, demonstrating that this inhibition is VDR dependent [Figure 2d]. Therefore, 1α, 25(OH)2D3 appeared to suppress NF-κB signaling, at least in part, by reducing IκBα and p65 NF-κB phosphorylation in HUVECs.

Figure 2: Effects of 1α,25-(OH)2D3 on the NF-κB signaling pathway. (a) Effect of NS50 on the expression of TNF-α-induced cell adhesion molecules (Western blotting). (b) Effect of NS50 on the expression of TNF-α-induced cell adhesion molecules (PCR). ✶ denotes P < 0.05 compared with cells treated with the blank control, # denotes P < 0.05 compared with cells treated with TNF-α, & denotes P < 0.05 compared with cells treated with TNF-α and SN50. (c) Effects of 1α,25-(OH)2D3 on NF-κB expression in HUVECs. (d) Effects of 1α,25-(OH)2D3 on NF-κB expression in HUVECs at different intervention times. ✶ denotes P < 0.05 compared with cells treated with the blank, # denotes P < 0.05 compared with cells induced with TNF-α, & denotes P < 0.05 compared with cells treated with 1α,25-(OH)2D3. TNF-α: Tumor necrosis factor-α, VDR-siRNA: Vitamin D receptor siRNA, 1α, 25(OH)2D3: 1α,25-dihydroxyvitamin D3, VCAM-1: Vascular cell adhesion molecule-1, PCR: Polymerase chain reaction, NF-κB: Nuclear factor kappa-B.

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Treatment of HUVECs with 1α,25-(OH)2D3 inhibits TNF-α-induced p65 NF-κB phosphorylation

Having confirmed that the NF-κB pathway is important for mediating VCAM-1 and E-selectin expression in HUVECs, we hypothesized that 1α, 25-(OH)2D3 might inhibit VCAM-1 and E-selectin expression by modulating the NF-κB pathway. To test this hypothesis, we examined the effects of 1α, 25-(OH)2D3 on IκBα and p65 NF-κB phosphorylation, p65 NF-κB expression, and p65 NF-κB binding to the VCAM-1 and E-selectin promoters. In other words, if p65 NF-κB could bind to the VCAM-1 and E-selectin promoters after 1α,25-(OH)2D3 treatment, then the presence of these two promoters could be determined. A ChIP assay demonstrated that 1α, 25-(OH)2D3 abrogated the binding of p65 NF-κB to its cognate cis-acting element in the VCAM-1 and E-selectin promoters in HUVECs. After cells were transfected with a specific VDR-siRNA, the inhibitory effect of 1α, 25-(OH)2D3 on p65 DNA binding was abrogated [Figure 3a]. This result indicates that 1α, 25(OH)2D3 blunts TNF-α-induced adhesion molecule expression by blocking NF-κB binding activity. Immunofluorescence staining of HUVEC with anti-p65 antibody showed that the expression of p65 NF-κB was significantly elevated in response to TNF-α stimulation. However, 1α, 25-(OH)2D3 reduced the expression of p65 NF-κB in HUVEC transfected with specific [Figure 3b]. Moreover, after the separation of the nucleus and cytoplasm, we again examined the expression of p65 and NF-κB. The results likewise revealed that intervention with TNF-α resulted in a significant increase in p65 NF-κB expression in the nucleus, which was significantly reversed after treatment with 1α, 25-(OH)2D3, and VDR-siRNA [Figure 3c].

Figure 3: Treatment of HUVECs with 1α,25-(OH)2D3 inhibits TNF-α-induced p65 NF-κB phosphorylation. (a) Results of ChIP analysis, ✶P < 0.05. (b) Effect of 1α,25-(OH)2D3 on p65 expression. (c) p65 NF-κB expression was detected after isolation of the nucleus and cytoplasm. ✶ denotes P < 0.05 compared with cells treated with the blank, # denotes P < 0.05 compared with cells treated with TNF-α, & denotes P < 0.05 compared with cells treated with 1α, 25-(OH)2D3 + TNF-α. TNF-α: Tumor necrosis factor-α, VDR-siRNA: Vitamin D receptor siRNA, 1α, 25(OH)2D3: 1α,25-dihydroxyvitamin D3, VCAM-1: Vascular cell adhesion molecule-1, DAPI: 4’,6-diamidino-2-phenylindole, ChIP: Chromatin immunoprecipitation, HUVECs: Human umbilical vein endothelial cells, PCR: Polymerase chain reaction, NF-κB: Nuclear factor kappa-B.

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Effect of 1α, 25-(OH)2D3 on the interaction between the VDR and p65 NF-κB

It has been reported that 1α, 25-(OH)2D3 can upregulate the expression of the VDR. Moreover, in human proximal tubular cells, vitamin D abolishes p65 NF-κB binding to the RANTES promoter by facilitating the VDR/p65 interaction. Our findings that the effects of 1α, 25-(OH)2D3 can be abrogated by a specific VDR-siRNA suggest that vitamin D exerts its beneficial effect at least partly through the VDR. Thus, we further explored the effects of 1α, 25-(OH)2D3 on VDR expression and the interaction between the VDR and p65 NF-κB in HUVECs. The results revealed that 1α, 25-(OH)2D3 upregulated the expression of the VDR in HUVECs [Figure 4a and b]. In addition, increased p65 expression was detected in the cell lysates precipitated with the anti-VDR antibody after stimulation with 1α, 25-dihydroxyvitamin D3 (or combined with TNF-α). However, p65 expression did not change under stimulation with TNF-α alone. We hypothesize that this is due to the increased p65 detected in the cell lysates precipitated by the anti-VDR antibody after stimulation with TNF-α and/or 1α,25-(OH)2D3 [Figure 4c]. Hence, increased formation of the VDR/p65 complex after 1α,25-(OH)2D3 treatment may reduce the level of free p65, thereby affecting its binding to the promoters of target genes and resulting in the inhibition of p65-mediated gene transcription.

Figure 4: Effects of 1α,25-(OH)2D3 on the interaction between the VDR and p65 NF-κB. a) Effects of 1α,25-(OH)2D3 on VDR expression in HUVECs (Western blotting). b) Effects of 1α,25-(OH)2D3 on VDR expression in HUVECs (PCR). ✶ denotes P < 0.05 compared with cells induced by TNF-α, # denotes P < 0.05 compared with cells treated with 1α,25-(OH)2D3. c) An immunoprecipitation assay was used to verify the effects of 1α,25-(OH)2D3 on p65 expression. ✶ denotes P < 0.05 compared with cells treated with the blank, # denotes P < 0.05 compared with cells treated with TNF-α and 1α, 25-(OH)2D3. TNF-α: Tumor necrosis factor-α, VDR: Vitamin D receptor, 1α, 25(OH)2D3: 1α,25-dihydroxyvitamin D3, VCAM-1: Vascular cell adhesion molecule-1, IP: Immunoprecipitation.

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DISCUSSION

This study demonstrated that 1α, 25-(OH)2D3, the active form of vitamin D, suppresses TNF-α-induced upregulation of VCAM-1 and E-selectin expression in HUVECs. Mechanically, 1α,25-(OH)2D3 targets the NF-κB signaling pathway, a principal signaling pathway involved in the regulation of inflammatory reactions in various circumstances, in a VDR-dependent manner. 1α, 25-(OH)2D3 can also upregulate the expression of VDR, promote the binding of VDR to p65, and thus inhibit the ability of p65 to bind to the VCAM-1 and E-selectin gene promoters. Since endothelial activation is a critical process that contributes to the pathogenesis of atherosclerosis and CV diseases, the inhibition of endothelial activation may be an important mechanism by which vitamin D ameliorates CV events.

As a fat-soluble ketosteroid, vitamin D is responsible for calcium and phosphorus metabolism and multiple other biological effects, including the regulation of the immune system and endothelial function. We have shown in our previous study that in CKD patients, hypovitaminosis D is associated with decreased brachial artery flow-mediated dilation (FMD) and increased VCAM-1 and E-selectin levels, and vitamin D supplementation can improve endothelial function.[20] The present study revealed that 1α, 25-(OH)2D3 suppresses TNF-α-induced high expression of VCAM-1 and E-selectin in HUVECs, which provides additional evidence for the endothelial protective effect of vitamin D through in vitro experiments. In agreement with our findings, Martinesi et al.[21] demonstrated that 1α, 25(OH)2D3 was able to reduce VCAM-1 levels previously increased by TNF-α; however, in their study, this effect was not observed on E-selectin expression, which might be explained by the experimental conditions. In fact, in their experiments, HUVECs were incubated with TNF-α in combination with 1α, 25(OH)2D3, and the two compounds were added at the same time.

Another issue worth mentioning is the intervention concentration of 1α, 25(OH)2D3. In this study, we pretreated HUVECs with various concentrations of 1α, 25-(OH)2D3 (10−9–10−7 M) and reported that 1α, 25(OH)2D3 at 10−8 M had the strongest inhibitory effect on TNF-α-induced adhesion molecule expression. Similarly, Kudo et al.[22] showed that in HCAECs, 1α, 25(OH)2D3 at a concentration of 10−8 M also had a slightly stronger suppressive effect on VCAM-1 expression after stimulation with TNF-α than did 1α, 25(OH)2D3 at concentrations of 10−7 M and 10−9 M. Moreover, Martinesi et al.[21] reported that in the absence of growth factors, 1α, 25(OH)2D3 at 10−8 M had a mildly stronger inhibitory effect on the expression of adhesion molecules in HUVECs than did 1α, 25(OH)2D3 at 10−7 M. Since hormones alone cannot alter the expression of adhesion molecules,[21] a relatively high concentration (10−7 M) may be needed for vitamin D to have severe inhibitory effects on HUVEC proliferation, as reported by Zehnder et al.,[23] leading to decreased expression of these molecules. Notably, 1α, 25(OH)2D3 at 10−7 M had a more obvious inhibitory effect on E-selectin expression and NF-κB activation than 10−8 M and 10−9 M 25(OH)2D3 in HCAECs.[16] Therefore, it is reasonable to use concentrations of 1α, 25-(OH)2D3 consistent with the levels obtained in healthy human plasma after the administration of a normal dose, which ranges from 10−9 to 10−7 M, as reported in the literature.[24,25]

The inhibition of NF-κB signaling with NF-κB SN50 reduced TNF-α-stimulated VCAM-1 and E-selectin expression in HUVECs, indicating that NF-κB signaling is required for inflammatory cytokine-induced endothelial activation. Previous investigations reported that the expression of NF-κB was greater in the vascular endothelial cells of subjects with vitamin D deficiency.[26] Hence, it is possible that vitamin D inhibits endothelial activation by inhibiting the NF-κB signaling pathway. The activities of NF-κB include IκBα and p65 NF-κB phosphorylation, p65 NF-κB expression, and p65 NF-κB binding to target gene promoters. Thus, the activity of NF-κB can be regulated at multiple stages. The present study demonstrated that 1α, 25-(OH)2D3 can affect p65 NF-κB binding to the VCAM-1 and E-selectin promoters. Although the exact mechanism by which vitamin D disrupts this interaction remains unclear, our data suggest that, in HUVECs, at least part of the mechanism stems from an increase in or stabilization of inhibitor of nuclear factor kappa-B α (IkBα), which reverses p65 expression. In agreement with our findings, several studies reported that in mesangial cells, pancreatic islet cells, and mouse embryonic fibroblasts, vitamin D can also inhibit NF-κB signaling through increasing IkBα and reducing p65 NF-κB expression, eventually abolishing its binding to gene promoters.[27-29] Therefore, more studies are needed to elucidate how vitamin D regulates IkBα.

As expected, in HUVECs transfected with a specific VDRsiRNA, the inhibitory effects of 1α, 25(OH)2D3 on VCAM-1 and E-selectin expression, as well as the NF-κB signaling pathway, were abolished, indicating that VDR is required to mediate the repressive effects of 1α, 25(OH)2D3. Moreover, 1α, 25-(OH)2D3 can upregulate the expression of VDR in HUVECs, which physically interacts with p65 and potentially blocks p65 binding to DNA.

Although our data strongly indicate that 1α, 25(OH)2D3 inhibits endothelial adhesion molecule expression primarily by triggering VDR-mediated inactivation of the NF-κB pathway, it remains to be elucidated whether this mechanism is applicable in vivo. In addition, 1α, 25(OH)2D3 may also regulate endothelial activation by other routes, given that vitamin D has pleiotropic effects.

This paper has been posted as a preprint on Research Square with DOI: 10.21203/rs.2.13428/v1, which is available from https://www.researchsquare.com/article/rs-4140/v1.[30]

SUMMARY

We have shown in this study that 1α, 25-(OH)2D3 can inhibit TNF-α-induced endothelial activation, and this potentially endothelial protective role seems to be mediated by its ability to induce the VDR-mediated inactivation of NF-κB signaling. In view of the importance of endothelial cell activation in the occurrence and development of CV events, 1α,25-(OH)2D3 may become a novel agent for the prevention and treatment of such diseases.

AVAILABILITY OF DATA AND MATERIALS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ABBREVIATIONS

1α, 25(OH)2D3: 1α, 25-Dihydroxyvitamin D3

ChIP: Chromatin immunoprecipitation

CKD: Chronic kidney disease

CV: Cardiovascular

DAPI: 4,6-diamidino-2-phenylindole

FITC: Fluorescein isothiocyanate

FMD: Flow-mediated dilation

HCAECs: Human coronary arterial endothelial cells

HUVECs: Human umbilical vein cells

SD: Standard deviation

TNF-α: Tumor necrosis factor-α

VCAM-1: Vascular cell adhesion molecule-1

VDR: Vitamin D receptor

AUTHOR CONTRIBUTIONS

QY.Z: Conceived and designed the project; YY.X and SX.L: Wrote and revised the manuscript; QY.S: Collected and analyzed data; Y.F: Visualization of the data; B.J: Supervised the study: YY.X and SX.L: Made equal contributions in this project as co-first authors. All authors read and approved the final submitted manuscript. All authors meet ICMJE authorship requirements.