ORIGINAL ARTICLE Year : 2021  |  Volume : 5  |  Issue : 3  |  Page : 140-147

Vitamin d-binding protein is involved in the pathogenesis of preeclampsia by inhibiting the tyrosine phosphorylation of vascular endothelial growth factor receptor-2 in endothelial cells

Ting-Feng Lu, Yun-Zhen Ye, Xiao-Tian Li, Ying Zhang
Department of Obstetrics, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200032, China

Date of Submission23-Feb-2021Date of Decision03-Mar-2021Date of Acceptance07-Jun-2021Date of Web Publication30-Jul-2021

Correspondence Address:
Ying Zhang
Room 903, Building 16, No. 888 Dong'an Road, Shanghai 200032
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.322839

Objective: The role of Vitamin D-binding protein (DBP) in preeclampsia (PE) pathogenesis is unknown. In this study, we compared the expression of DBP in the placentas of PE patients with the placentas of normotensive pregnant women with placenta previa controls, and aimed to explore the effect of DBP on endothelial cells (ECs) and the underlying mechanism.
Methods: DBP expression in placental tissues collected from PE patients and controls was evaluated by immunohistochemistry. The downregulation and upregulation of DBP expression in HTR-8/SVneo cells were examined using DBP-targeting small interfering RNA (siRNA) and DBP-expression vector, respectively. The conditioned media of these DBP-overexpressing and DBP-siRNA HTR-8/SVneo cells were collected and added to human umbilical vein EC (HUVEC) cultures. Angiogenic effects on HUVECs were assessed by tube formation assays, and the proliferation and migration of HUVECs were examined using the Real-Time Cell Analyzer. The expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2, as well as the phosphorylation of different residues of VEGFR-2 in HUVECs, were determined by western blotting.
Results: DBP expression was significantly increased in the placental tissues collected from PE patients. The conditioned medium of DBP-overexpressing HTR-8/SVneo cells potently inhibited tube formation by HUVECs, in addition to their proliferation and migration. Furthermore, treatment of HUVECs with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells decreased the phosphorylation of VEGFR-2 at tyrosine 996, whereas the treatment of these cells with the conditioned medium of DBP-siRNA HTR-8/SVneo cells increased the phosphorylation of VEGFR-2 at tyrosine 951, 996, and 1,175.
Conclusions: The expression of DBP is increased in the placentas of PE patients. DBP plays potential roles in endothelial dysfunction, which contributes to PE development, by inhibiting tyrosine phosphorylation of VEGFR-2 in ECs.

Keywords: Angiogenesis; Phosphorylation; Preeclampsia; Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor-2; Vitamin D-Binding Protein

How to cite this article:
Lu TF, Ye YZ, Li XT, Zhang Y. Vitamin d-binding protein is involved in the pathogenesis of preeclampsia by inhibiting the tyrosine phosphorylation of vascular endothelial growth factor receptor-2 in endothelial cells. Reprod Dev Med 2021;5:140-7
How to cite this URL:
Lu TF, Ye YZ, Li XT, Zhang Y. Vitamin d-binding protein is involved in the pathogenesis of preeclampsia by inhibiting the tyrosine phosphorylation of vascular endothelial growth factor receptor-2 in endothelial cells. Reprod Dev Med [serial online] 2021 [cited 2021 Sep 19];5:140-7. Available from: https://www.repdevmed.org/text.asp?2021/5/3/140/322839   Introduction 

Preeclampsia (PE), characterized by hypertension, proteinuria, and maternal multiorgan dysfunction, is one of the most common pregnancy-specific complications. It affects approximately 4.6% (range: 2.7%–8.2%) of all pregnant women.[1] PE induces maternal and perinatal complications and remains a major cause of maternal and neonatal mortality.[2] Moreover, PE predisposes women to cardiovascular diseases beyond their pregnancies.[3] However, the precise etiology and pathogenesis of PE remain unclear.

Successful pregnancy depends on sophisticated angiogenesis and vasculogenesis to meet the developmental requirements of the fetus. Although the precise pathophysiology of PE is still unclear, it is generally recognized that abnormal placentation and aberrant vascularization may contribute to PE occurrence. Maternal vascular endothelial dysfunction and vascular lesions can induce hypertensive phenotypes and potentially fatal complications of vital organs, such as kidney failure, eclampsia, stroke, liver rupture, and pulmonary edema.[4] In addition, fetal growth restriction is associated with uteroplacental dysfunction.

Vascular endothelial growth factor (VEGF) is a major angiogenic factor that interacts with VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2)/kinase insert domain receptor in endothelial cells (ECs). It can regulate the proliferation, differentiation, migration, vasculogenesis, and vascular permeability of ECs. VEGF has been reported to increase the concentration of intracellular free calcium ([Ca2+]i) in ECs, enhance endothelial nitric oxide synthase (eNOS) activity, and promote Ca2+/calmodulin and eNO production, which lead to decreased vessel tonicity and blood pressure.[5] VEGF, through its receptors, is also thought to play an active role in normal vascular adaptation to placentation[6] and the regulation of trophoblast function. In nonpregnant women, inhibition of VEGF signaling following treatment with bevacizumab (a VEGF-neutralizing antibody) induces PE symptoms, such as hypertension, proteinuria, and elevation of liver enzyme levels.[7] Conversely, treatment with adenosine monophosphate-activated protein kinase increases VEGF expression, which ameliorates hypertension in rats with reduced uteroplacental perfusion pressure.[8] Notably, women with PE have decreased levels of VEGF, VEGFR-1, and VEGFR-2 in the peripheral blood.[9],[10]

Vitamin D-binding protein (DBP) is a sparsely glycosylated (0.5%–1.0%) α2-globulin, which belongs to the albumin superfamily and is expressed in the placenta, uterus, heart, brain, lungs, kidneys, and spleen as well as on the surfaces of lymphocytes, monocytes, and neutrophils.[11] The main function of DBP is to bind, solubilize, and transport Vitamin D to target organs and cells. The active form of Vitamin D (1,25[OH] 2D3) can bind to the Vitamin D receptor, which promotes VEGF transcription.[12] Increased secretion of VEGF facilitates the proliferation of vascular smooth muscle cells in vitro.[13] Moreover, DBP is known to perform numerous important biological functions. Following treatment with β-galactosidase and sialidase, DBP can be converted to DBP macrophage-activating factor (DBP-MAF). The latter exhibits direct anti-angiogenic effects on human ECs (HECs) by blocking ERK1/2 and VEGFR-2 signaling cascades, which inhibit EC proliferation, chemotaxis, migration, tube formation, and capillary sprouting.[14],[15],[16] However, the relationship between DBP and VEGF expression in PE pathogenesis has not been adequately elucidated.

In this study, we investigated the role of DBP in PE pathogenesis. We found that DBP expression was notably elevated in the placentas of patients with PE compared with placentas of the normotensive pregnant women with placenta previa. We demonstrated that the treatment of human umbilical vein ECs (HUVECs) with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells (simulating high DBP levels in the placental microenvironment) inhibited their angiogenesis, proliferation, and migration. In contrast, the treatment of HUVECs with the conditioned medium of DBP-small interfering RNA (siRNA) HTR-8/SVneo cells promoted angiogenesis. Although the total VEGF/VEGFR-2 expression remained unchanged, the phosphorylation of VEGFR-2 at tyrosine 996 in HUVECs treated with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells was significantly decreased, whereas the phosphorylation of VEGFR-2 at tyrosine 951, 996, and 1,175 was increased in HUVECs treated with the conditioned medium of DBP-siRNA HTR-8/SVneo cells.

  Methods 

Placental tissue collection

Placental tissues were collected at the time of delivery by cesarean section from women with PE (33–36 weeks, n = 13) before active labor. Gestational age-matched placentas (n = 11) were obtained from normotensive pregnant women (control) with placenta previa. [Table 1] presents the clinical details of the study groups. Maternal age, gestational age, parity, and mode of delivery were not significantly different between the two groups. As expected, systolic and diastolic blood pressure and urinary protein levels were significantly higher in the PE group than in the control group. The weight of newborns in the PE group was decreased. None of the participants had any other complications.

Fresh placental tissue samples measuring 1 cm × 1 cm were washed with sterile phosphate-buffered saline (PBS), fixed in 10% buffered formalin, embedded in paraffin for sectioning, and further processed for immunohistochemistry (IHC). This study was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University, and written informed consent was obtained from all participants.

Placental immunohistochemistry for Vitamin D-binding protein

DBP expression was examined using a standard IHC staining procedure. Placental samples were sliced into 5-μm-thick sections, deparaffinized, rehydrated, and processed for wet heat-induced antigen retrieval. Placental tissue sections were blocked for 1 h at room temperature (20°C–25°C) with 1% bovine serum albumin in PBS and incubated with the primary antibody against DBP (1:100, Abcam, Cambridge, UK) overnight at 4°C, followed by HRP-conjugated secondary antibody for 1 h at room temperature. The slides were counterstained with Harris hematoxylin (Sigma-Aldrich, MO, USA). Immunostaining intensity and area were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, MD, USA).

HTR-8/SVneo cell culture and transfection with Vitamin D-binding protein-small interfering RNA or Vitamin D-binding protein expression plasmid

The HTR-8/SVneo cells (immortalized trophoblast cell line) were cultured on plates containing DMEM/F12 (HyClone, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, CA, USA), 200 mmol/L L-glutamine, and antibiotics. The cells were cultured in a humidified 5% CO2 incubator at 37°C and grown in a monolayer. The cells were trypsinized with 0.25% Trypsin-EDTA (HyClone) and passaged every 3 days.

Silencing of the DBP gene in HTR-8/SVneo cells was performed using DBP-targeting small interfering RNA (DBP-siRNA) and Lipofectamine 2000 (Life Technologies, CA, USA) according to the manufacturer's protocol. The corresponding siRNA sequences were designed by GenePharma (Shanghai, China) and are presented in [Supplementary Table 1]. The scrambled siRNA was used as a negative control siRNA (NC siRNA). Briefly, HTR-8/SVneo cells were seeded onto a six-well culture plate, and when the cells reached 60%–70% confluence, they were transfected with 5 μL Lipofectamine 2000 and 5 μL DBP-siRNA or scrambled siRNA in Opti-MEM reduced serum medium in the absence of antibiotics for 24 h. After transfection for 24 h, the culture medium was replaced with DMEM/F12 supplemented with 10% FBS and antibiotics.

DBP was overexpressed in HTR-8/SVneo cells using the DBP expression vector (DBP-vector). The pcDNA3.1+-DBP plasmid was designed and produced by GenePharma (Shanghai, China). The corresponding plasmid sequences are shown in [Supplementary Figure 1]. Briefly, the full-length cDNA sequence of human DBP was cloned and subcloned to the pcDNA3.1+ vector using DNA ligase. Subsequently, subcloning was confirmed by sequencing. The pcDNA3.1+ vector was used as a negative control vector (NC vector). HTR-8/SVneo cells were seeded onto a six-well culture plate, and when the cells reached 80%–90% confluence, they were transfected with 10 μL Lipofectamine 2000 and 10 μL pcDNA3.1+-DBP plasmid or the pcDNA3.1+ plasmid in Opti-MEM reduced serum medium in the absence of antibiotics. After incubation for 5 h, the culture medium was replaced with DMEM/F12 supplemented with 10% FBS and antibiotics. Cells were collected after 48 h for transfection efficiency analysis by detecting DBP expression at the mRNA and protein levels. The conditioned media of HTR-8/SVneo cells were collected after 48 h of transfection and immediately stored at −80°C.

Figure 1: Expression of Vitamin D-binding protein in human placental tissues. (a) Immunostaining for Vitamin D-binding protein in placentas from PE patients and matched controls. Representative photographs at ×10 (above) and ×40 (below) magnifications. (b) Quantitative analysis of the mean density of immunostaining using Image-Pro Plus 6.0. Data are expressed as mean ± SEM (13 cases of PE and 11 cases of CTRL). *P < 0.001. CTRL: Matched control; PE: Preeclampsia; SEM: Standard error of the mean.

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Quantitative reverse transcription-polymerase chain reaction

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the transfection efficiency. HTR-8/SVneo cells were collected 48 h after transfection, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). qRT-PCR was conducted using a cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) to synthesize cDNA. qRT-PCR was performed using the ABI 7900 HT Real-Time PCR System (Thermo Fisher Scientific) using the SYBR green detection method (Takara, Japan) according to the manufacturer's protocol. GAPDH was used as an internal reference. The primer sequences for DBP and GAPDH are listed in [Supplementary Table 2]. The melting curve analysis was performed to verify amplification specificity.

Isolation and culture of human umbilical vein endothelial cells

HUVECs were isolated from human umbilical cords. Briefly, human umbilical cords were collected at the time of delivery by cesarean section (gestational age of 37–39 weeks) without any complications. Umbilical veins were isolated, washed three times with cold PBS containing 1% penicillin and 1% streptomycin, infused with DMEM/F12 (HyClone) containing 1% collagen I, incubated for 20 min in a 5% CO2 incubator at 37°C, and then washed again with PBS. The mixture containing the vein samples and PBS was then centrifuged at 200 ×g for 10 min. The supernatant was discarded, and HUVECs were resuspended and cultured in ECM medium (ScienCell, CA, USA) containing 5% FBS and 1% endothelial growth factor in a 5% CO2 incubator at 37°C. HUVECs were trypsinized with 0.25% Trypsin-EDTA (HyClone) and passaged every 3 days. HUVECs passaged for 3–5 times were used for further experiments.

Human umbilical vein endothelial cell tube formation

HUVECs grown in complete endothelial growth medium were seeded onto a growth factor-free Matrigel-coated (Corning, NY, USA) 96-well plate and treated with the conditioned medium of HTR-8/SVneo cells transfected with DBP-siRNA or pcDNA3.1+-DBP plasmid at a 1:1 ratio with complete medium. After incubation for 4 h in a 5% CO2 incubator at 37°C, the cells demonstrated tube formation and were photographed. The number and size of the tubes were calculated using Image-Pro Plus 6.0 (Media Cybernetics, MD, USA).

Human umbilical vein endothelial cell proliferation and migration

HUVEC proliferation and migration were examined using the Real-Time Cell Analyzer (RTCA; ROCHE, Basel, Switzerland) according to the manufacturer's protocols. Briefly, 2,000 cells were added to the E-plate or CIM-plate, and complete medium was removed when the cells were adherent to the plate. Subsequently, the conditioned medium of HTR-8/SVneo cells transfected with DBP-siRNA or pcDNA3.1+-DBP plasmid at a 1:1 ratio with complete medium was added. The cells were then cultured for 120 min at 37°C. The cell indices were recorded using the RTCA.

Western blotting

Western blotting was performed to determine whether VEGF, VEGFR-2, and phosphorylated VEGFR-2 expression levels in HUVECs were altered following treatment with the conditioned medium of HTR-8/SVneo cells transfected with DBP-siRNA or pcDNA3.1+-DBP plasmid. HUVECs were incubated with the conditioned medium of HTR-8/SVneo cells transfected with DBP-siRNA or pcDNA3.1+-DBP plasmid at a 1:1 ratio with complete medium and collected after 48 h. Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) and PMSF/phosphatase inhibitors (Beyotime) at a 100:1 ratio. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime). Next, 10–20 μg protein was loaded and separated on a 10% polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica MA, USA). The blotted membranes were incubated with 5% evaporated milk for 1 h at room temperature. Primary antibodies against DBP (Abcam, Cambridge, UK; ab81307), VEGF (Abcam, ab32152), VEGFR-2 (Abcam, ab221679), GAPDH (Abcam, ab8245), VEGFR-2-P951 (Abcam, ab38473), VEGFR-2-P996 (Invitrogen, PA5-105765), and VEGFR-2-P1175 (Abcam, ab194806) were diluted at a 1:500 ratio and incubated with the corresponding membranes overnight at 4°C. The membranes were then incubated with secondary antibodies (Abcam), which were diluted at a 1:10,000 ratio, at room temperature for 1 h. The protein bands were visualized using a Chemiluminescence Kit (Millipore), and the gray values were analyzed using ImageJ (Rawak Software, Stuttgart, Germany).

Statistical analysis

All experiments were repeated at least three times. Data were analyzed using the software GraphPad Prism version 5 (GraphPad Software, CA, USA). One-way analysis of variance or Student's t-test was used to evaluate differences between multiple groups or two groups. All data are expressed as the mean ± standard error of the mean. Statistical significance was set at P < 0.05.

  Results 

Vitamin D-binding protein expression is increased in preeclampsia placental tissues

Placental tissues from patients with PE and controls were collected, and IHC was performed to detect DBP expression. As shown in [Figure 1]a, the expression of DBP was low in the placental tissues of normotensive controls, mainly in trophoblasts, including cytotrophoblasts and syncytiotrophoblasts. However, in the placental tissues of PE patients, DBP expression, in both cytotrophoblasts and syncytiotrophoblasts, was markedly higher than that in the placental tissues of normotensive controls (P < 0.001) [Figure 1]a and [Figure 1]b. The increased expression of DBP in PE placentas suggests a role of DBP in the occurrence or progression of PE.

Conditioned medium of Vitamin D-binding protein-overexpressing HTR-8/SVneo cells impedes tube formation in human umbilical vein endothelial cells

To clarify whether DBP is involved in the development of PE, we next explored the effect of DBP on trophoblasts and ECs, which are the major components of the placenta. HTR-8/SVneo cells (trophoblast cell line) were transfected with DBP-siRNA or DBP-expression vector to downregulate or upregulate DBP expression, respectively. Transfection efficiency was evaluated using qRT-PCR [Supplementary Figure 2] and western blotting [Figure 2]a. The conditioned medium of HTR-8/SVneo cells, as described above, was added to the HUVEC culture system. As shown in [Figure 3], when treated with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells, a significantly diminished formation of capillary-like structures by HUVECs was detected. In contrast, tube formation by HUVECs was markedly promoted after treatment with the conditioned medium of DBP-siRNA HTR-8/SVneo cells. These data suggest that DBP could play a negative role in the placental vascular system by inhibiting angiogenesis.

Figure 2: VEGF and VEGFR-2 protein expression in HUVECs incubated with conditioned medium from HTR-8/SVneo cells transfected with DBP-siRNA, pcDNA3.1+-DBP plasmid, or the respective controls. (a) DBP expression levels in HTR-8/SVneo cells were detected using western blot. (b) Representative photographs of VEGF and VEGFR-2 protein expression in HUVECs. (c and d) Statistical analysis of VEGF and VEGFR-2 protein expression in HUVECs (n = 3). No significant difference in VEGF and VEGFR-2 expression was observed among the groups. Data are expressed as means ± SEM. CTRL (non-treated HTR-8/SVneo), NC-vector (scramble vector-transfected HTR-8/SVneo), NC-siRNA (NC siRNA-transfected HTR-8/SVneo), DBP-vector (DBP expression vector-transfected HTR-8/SVneo), DBP-siRNA (DBP siRNA-transfected HTR-8/SVneo). HUVECs: Human umbilical vein endothelial cells; CTRL: Matched control; SEM: Standard error of the mean; VEGFR-2: Vascular endothelial growth factor receptor-2; VEGF: Vascular endothelial growth factor; siRNA: Small interfering RNA; DBP: Vitamin D-binding protein.

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Figure 3: Conditioned medium of DBP-overexpressing HTR-8/SVneo cells impedes tube formation in HUVECs. HUVECs (5,000 cells) were seeded onto Matrigel-precoated 96-well plates. Conditioned medium collected from HTR-8/SVneo cells transfected with DBP-siRNA, pcDNA3.1+-DBP plasmid or the respective controls were added to the HUVEC cultures. Tube formation was observed and photographed after 4 hours. Representative photographs (a) and statistical analysis (b) of tube formation in cultures treated as above. Data are expressed as mean ± SEM. *P < 0.05, †P < 0.01. CTRL (non-treated HTR-8/SVneo), NC-vector (scramble vector-transfected HTR-8/SVneo), NC-siRNA (NC siRNA-transfected HTR-8/SVneo), DBP-vector (DBP expression vector-transfected HTR-8/SVneo), DBP-siRNA (DBP siRNA-transfected HTR-8/SVneo). HUVECs: Human umbilical vein endothelial cells; SEM: Standard error of the mean; CTRL: Matched control; siRNA: Small interfering RNA; DBP: Vitamin D-binding protein.

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Conditioned medium of Vitamin D-binding protein-overexpressing HTR-8/SVneo cells suppresses the proliferation and migration of human umbilical vein endothelial cells

RTCA was used to quantify the effect of DBP on the proliferation and migration of HUVECs. As depicted in [Figure 4]a, HUVECs treated with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells showed significantly diminished proliferation (n = 3, P < 0.01). Nevertheless, the conditioned medium of DBP-siRNA HTR-8/SVneo cells had no noticeable effect on HUVEC proliferation. In addition, our results demonstrated that the migration of HUVECs was decreased in the group supplemented with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells. Interestingly, the conditioned medium of DBP-siRNA HTR-8/SVneo cells did not affect HUVEC migration [n = 3, P < 0.05, [Figure 4]b. Collectively, these results suggest that DBP inhibits the proliferation and migration of HUVECs.

Figure 4: Conditioned medium of DBP-overexpressing HTR-8/SVneo cells suppresses the proliferation and migration of HUVECs. (a) Approximately 2,000 HUVECs/well were cultured in E-plates in complete media. After HUVECs adhered to the plate (at the platform), the medium was removed and cells were incubated with conditioned medium from HTR-8/SVneo cells transfected with DBP-siRNA, pcDNA3.1+-DBP plasmid, or the respective controls. The proliferation of HUVECs was significantly reduced in the group treated with conditioned medium of the DBP-vector group (†P < 0.01). Comparable proliferation of HUVECs was observed in the other four groups. (b) 2,000 cells/well were seeded onto a CIM-plate and then inserted into the wells containing complete medium. HUVECs were pretreated for 20 min with the conditioned medium from HTR-8/SVneo cells transfected with DBP-siRNA, pcDNA3.1+-DBP plasmid, or the respective controls. Migration of HUVECs was significantly reduced in the group treated with the conditioned medium of the DBP-vector group (*P < 0.05). Comparable migration of HUVECs was observed in the other four groups. Data are expressed as mean ± SEM. CTRL (nontreated HTR-8/SVneo), NC-vector (scramble vector-transfected HTR-8/SVneo), NC-siRNA (NC siRNA-transfected HTR-8/SVneo), DBP-vector (DBP expression vector-transfected HTR-8/SVneo), DBP-siRNA (DBP siRNA-transfected HTR-8/SVneo). HUVECs: Human umbilical vein endothelial cells; SEM: Standard error of the mean; CTRL: Matched control; siRNA: Small interfering RNA; DBP: Vitamin D-binding protein.

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Conditioned medium of Vitamin D-binding protein-overexpressing HTR-8/SVneo cells inhibits the phosphorylation of vascular endothelial growth factor receptor-2 and blocks vascular endothelial growth factor/vascular endothelial growth factor receptor-2 signaling in human umbilical vein endothelial cells

To elucidate the role of DBP in modulating the biological function of HUVECs, we examined the effect of DBP on VEGF/VEGFR-2 signaling. The expression of VEGF and VEGFR-2 was examined in HUVECs treated with conditioned media from HTR8/SVneo cells transfected with DBP-siRNA or pcDNA3.1+-DBP plasmid. As shown in [Figure 2]b, [Figure 2]c, [Figure 2]d, there was no significant difference in the expression of VEGF and VEGFR-2 at the protein level among the different treatment groups. Next, we examined the phosphorylation of VEGFR-2 at different sites. Notably, the treatment of HUVECs with the conditioned medium of DBP-siRNA cells, increased the phosphorylation of VEGFR-2 at tyrosine 951, 996, and 1,175 significantly (P < 0.05, P < 0.01). In contrast, the treatment of HUVECs with the conditioned medium of DBP-overexpressing cells resulted in significantly decreased phosphorylation of VEGFR-2 at tyrosine 996 (P < 0.01) [Figure 5]. Our results suggest that DBP inhibits the phosphorylation of VEGFR-2 at certain sites in HUVECs, which may further interfere with the VEGF/VEGFR-2 signaling pathway involved in the normal functioning of HUVECs in the placenta.

Figure 5: Conditioned medium of DBP-overexpressing HTR-8/SVneo cells inhibits the phosphorylation of VEGFR-2 in HUVECs. Representative photograph (a) and quantitative analysis (b-d) of phosphorylation of VEGFR-2 at different sites (tyrosine 951, 996 and 1,175) in HUVECs by western blots (n = 3). Significantly increased phosphorylation levels at tyrosine 951, 996, and 1,175 of VEGFR-2 were detected in the DBP-siRNA group compared to those in the NC-siRNA group. The VEGFR-2 tyrosine 996 phosphorylation was notably inhibited in the DBP-vector group compared with that in the NC-vector group. *P < 0.05, †P < 0.01. Data are expressed as means ± SEM. CTRL (non-treated HTR-8/SVneo), NC-vector (scramble vector-transfected HTR-8/SVneo), NC-siRNA (NC siRNA-transfected HTR-8/SVneo), DBP-vector (DBP expression vector-transfected HTR-8/SVneo), DBP-siRNA (DBP siRNA-transfected HTR-8/SVneo). HUVECs: Human umbilical vein endothelial cells; CTRL: Matched control; SEM: Standard error of the mean; VEGFR-2: Vascular endothelial growth factor receptor-2; siRNA: Small interfering RNA; DBP: Vitamin D-binding protein.

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  Discussion 

Despite the numerous studies and remarkable progress in research and therapy, the physiopathology and molecular mechanisms underlying the development of PE remain largely unknown. The pathogenesis of PE is associated with maternal endothelial dysfunction. In this study, we found that DBP expression was significantly upregulated in preeclamptic placentas. In addition, we demonstrated that upregulation of DBP expression in trophoblast cells inhibited tube formation, proliferation, and migration of HUVECs in vitro. Furthermore, we found that DBP did not alter the expression levels of VEGF and VEGFR-2, but it controlled the phosphorylation of VEGFR-2. These findings suggest that high expression levels of DBP in preeclamptic placentas may lead to endothelial vascular dysfunction by inhibiting VEGFR-2 phosphorylation.

DBP, the major binding protein for 25(OH) D and 1,25(OH)2D, a key biomolecule regulating Vitamin D homeostasis, also serves as an immunomodulator during successful pregnancy. It has been verified that serum concentrations of DBP increase during pregnancy.[17],[18] However, dysregulation of DBP equilibrium has also been suggested to contribute to the pathogenesis of PE. Kolialexi et al. found that plasma DBP expression in the first trimester of pregnancy was notably upregulated in women who subsequently developed early-onset PE compared with that in women with a normal pregnancy.[19] Consistent with the results of a previous study, we confirmed the increased expression levels of DBP in preeclamptic placentas in this study. As DBP expression was upregulated in the placenta and plasma of PE patients, we hypothesized that increased DBP expression in the preeclamptic placenta is a potential source of DBP in maternal circulation. The expression of DBP in PE remains controversial. Powe et al. revealed that serum DBP was not differentially expressed in the first trimester between women with PE and women with a normal pregnancy, also, the serum DBP was not associated with blood pressure in the first trimester.[20] Ma et al. reported that DBP expression was reduced in syncytiotrophoblasts in preeclamptic placentas. Another study showed no significant difference in the protein levels of DBP between placentas of women with PE and women with a normal pregnancy.[21] The reasons underlying the inconsistencies in DBP expression in PE remain unclear. However, the inconsistencies can potentially be attributed to the different DBP measurement methods,[22] small sample size, antibody specificity, and tissue collection pattern.[23]

DBP-MAF, formed by the combined action of β-galactosidase and sialidase on DBP, has been shown to inhibit proliferation, migration, tube formation, and microvessel sprouting of HECs.[24],[25] However, the direct function of DBP in angiogenesis has rarely been examined. In the present study, we found that the conditioned medium of DBP-overexpressing HTR-8/SVneo cells (simulating high DBP levels in the placental microenvironment) could inhibit proliferation, migration, and tube formation in HUVECs, whereas the conditioned medium of DBP-siRNA HTR-8/SVneo cells was found to promote angiogenesis. These findings indicate that excessive DBP levels in the placenta result in poor vascularization related to PE.

In the endothelium, VEGF and VEGFR-2, with potent tyrosine kinase activity, induce the major crucial angiogenic signaling pathways.[2],[6] Furthermore, they are implicated in both placental and fetal angiogenesis. Tyrosine phosphorylation of VEGFR-2 subsequently triggers multiple downstream signaling pathways to induce angiogenesis of ECs,[27] including FAK, PI3K, AKT, mTOR, MAPK, and ERK pathways.[28],[29] In particular, tyrosines 951, 996, and 1,175 of VEGFR-2 are rapidly phosphorylated following VEGF binding resulting in the activation of VEGFR-2, which affects the proliferation, migration, and angiogenesis of ECs.[30] In this study, we found that the phosphorylation of tyrosines 951, 996, and 1,175 of VEGFR-2 was concurrently enhanced in the DBP-siRNA group, while only the phosphorylation of tyrosine 996 of VEGFR-2 was inhibited in the DBP-vector group. We also demonstrated that the conditioned medium of DBP overexpressing HTR-8/SVneo cells could efficiently affect HUVEC proliferation, migration, and tube formation, whereas the conditioned medium of DBP-siRNA HTR-8/SVneo cells had an impact on angiogenesis only. Therefore, we hypothesized that higher DBP levels impede the proliferation, migration, and angiogenesis of ECs by inhibiting the phosphorylation of tyrosine 996 of VEGFR-2 in PE. However, downregulation of DBP promoted tube formation in HUVECs, despite its role in stimulating the phosphorylation of tyrosine 951, 996, and 1,175 of VEGFR-2. We assume that some other factors co-exist, which offset the effects of phosphorylated VEGFR-2 on cell proliferation and migration induced by the aberrant expression of DBP. Thus, our study suggests one of the mechanisms by which DBP plays a role in PE pathogenesis.

A limitation of this study was that only 13 patients were included in the PE group. Future studies with a larger sample cohort are needed to identify a clear association between DBP expression levels and PE. In addition, we used the conditioned medium of HTR-8/SVneo cells overexpressing DBP to simulate high DBP expression in the PE placenta milieu; however, the exact source of the high DBP in PE remains to be elucidated. The detailed roles of VEGFR-2 and DBP, which are critical for the proliferation, migration, tube formation, and actin filament formation in HUVECs,[31] should also be further explored in the context of PE.

In summary, the present study revealed that DBP expression is upregulated in preeclamptic placentas. Moreover, increased DBP expression levels altered HUVEC proliferation, migration, and tube formation, potentially via inhibition of the tyrosine phosphorylation of VEGFR-2. Although clinical applications require further investigation, this study provides the experimental basis for examining the important role of upregulated DBP expression in aberrant angiogenesis in PE.

Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.

Acknowledgments

We are grateful to the pregnant women who participated in this study.

Financial support and sponsorship

This study was supported by the National Key R and D Program of China (2016YFC1000403) and the National Nature Science Foundation of China (81601311 and 81300491).

Conflicts of interest

There are no conflicts of interest.

 

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
  [Table 1]