Inhibition of Wnt Inhibitory Factor 1 Under Hypoxic Condition in Human Umbilical Vein Endothelial Cells Promoted Angiogenesis in Vitro

Abstract

Placentation is a complicated process critical for maternal–fetal exchange of nutrients and gases that includes stepwise vasculogenesis and angiogenesis. Wnt inhibitory factor 1 (WIF1) is a secreted Wnt antagonist that acts as a tumor-suppressor gene by antagonizing angiogenesis and proliferation and inducing apoptosis. The purpose of this study was to investigate the function of WIF1 on placental angiogenesis in human umbilical vein endothelial cells (HUVECs) under hypoxic conditions. We found that WIF1 was diversely expressed in placental vascular endothelial cells at different points during gestation and was weaker in the early placenta than in the term placenta. We validated the antiangiogenesis role of WIF1 by inhibiting proliferation, tube formation and migration, and inducing apoptosis of endothelial cells through antagonizing Wnt/β-catenin signaling pathway. We also identified that hypoxic conditions similar to the early placenta inhibited the expression of WIF1 and reversed the antiangiogenesis of WIF1 in HUVECs. In conclusion, our present study supported the hypothesis that WIF1 is crucial as a negative regulator of the functions of endothelial cells in angiogenesis and that hypoxia plays an important role in controlling WIF1 expression and angiogenesis. We also demonstrated that Wnt/β-catenin signaling pathway was activated in correspondence with the suppression of WIF1 in the angiogenesis of endothelial cells under hypoxic conditions.

Keywords

hypoxia angiogenesis Wnt inhibitory factor 1 human umbilical vein endothelial cells

Introduction

In human pregnancy, successful placentation is required for the exchange of nutrients, oxygen, and waste between the mother and the developing fetus. Proper placental development involves the processes of vasculogenesis (de novo vessel formation of capillaries) and angiogenesis (sprouting, bridging, and intussusceptive growth from existing vessels) occurring stepwise to form contractile vessels during early gestation.¹ During this process, endovascular extravillous trophoblasts (EVTs) replace the maternal endothelial cells (ECs),^2
–4 and, subsequently, endothelial precursor cells sprout to form the vascular network. Then, the maternal spiral arterioles transform from high-resistance, low-flow muscular vessels into low-resistance, high-flow sac-like vessels allowing adequate maternal blood flow to the placenta.⁵ Disordered vasculogenesis and angiogenesis contribute to impaired placentation and the development of pathological pregnancies, such as abortion, preeclampsia, and intrauterine growth restriction.

Several factors have been identified that control placental angiogenesis, including intrinsic factors secreted from the EVT,^6,7 paracrine decidual factors, growth factors, cytokines,^8,9 and microenvironmental elements, such as oxygen concentration.¹⁰ During the first 8 to 12 weeks of gestation, EVT plugs block the spiral arteries causing the partial pressure of oxygen in the placenta to become significantly lower than the endometrium, thereby creating an environment of physiological hypoxia for placental angiogenesis. Moreover, hypoxia is essential for angiogenesis in wound healing, fetal development, and tumor formation. The oxygen-sensitive transcriptional activator hypoxia-inducible factor 1 (HIF-1) is a key transcriptional mediator in the response to hypoxic conditions.¹¹ A large number of proangiogenic growth factors, such as vascular endothelial growth factor (VEGF)^12
–14 of the HIF pathway, induce many of the individual phenotypic processes in angiogenesis such as migration or tube formation of ECs in response to hypoxia.¹⁵

Wnts are a family of 19 secreted, cysteine-rich glycoproteins in human that signal through the canonical Wnt/β-catenin pathway and the noncanonical Wnt/calcium and planar cell polarity pathways, activating several developmental signaling pathways.¹⁶ Activation of the canonical Wnt pathway features β-catenin dephosphorylation and then enables β-catenin translocation to the nucleus and gene transcription modulation.¹⁷ Numerous genes controlling vascular developmental processes related to cell proliferation, adhesion, and migration such as cyclin D1 and VEGF¹⁸ are transcriptionally regulated by the Wnt signaling pathway. Endothelial-specific deletion of β-catenin causes a defective vascular pattern and increased vascular fragility.¹⁹ Several Wnts, such as Wnt1, Wnt2, Wnt7b, and Wnt4, can induce canonical β-catenin activation and regulate various aspects of EC behavior during vascular system morphogenesis.^20

–23 The noncanonical Wnt pathway activated by Wnt5a is also involved in placental and embryonic vascular development.²⁴ Wnt signaling antagonists, such as secreted frizzled-related proteins, are involved in endothelial signaling.²⁵

Wnt inhibitory factor 1 (WIF1) is a secreted Wnt antagonist that directly interacts with Wnt ligands abrogating their downstream signal. In general, WIF1 is considered a tumor-suppressor gene because it suppresses invasion and induces apoptosis in cancer cell lines.^26,27 In vivo and in vitro researches also indicate its role in angiogenesis antagonism. Wnt inhibitory factor 1 interacts with Wnt1, prevents Wnt/β-catenin activation, and exerts antiangiogenic effects through VEGF downregulation in cancer cells.²⁸ Additionally, WIF1 strongly inhibited the formation of hepatic sinusoidal ECs on matrigel.²¹

However, few data are available on the function of WIF1 in HUVECs under hypoxic conditions in placental angiogenesis. Therefore, we utilized a lentivirus overexpressing WIF1 gene to investigate the effects of WIF1 on EC migration and tube formation in vitro. We also cultured HUVECs under hypoxic conditions to mimic the hypoxic environment of the early placenta and further explore the influence of hypoxia on WIF1 in angiogenesis.

Materials and Methods

Tissue Collection

Human first trimester placental (n = 10) and decidual tissues (n = 10; 8-10 weeks) were obtained from healthy women undergoing legal abortion for nonmedical reasons. The normal term placenta tissues (n = 10) were collected from women undergoing cesarean sections without labor at 37 to 40 weeks of pregnancy without any complications. Informed consent was obtained from all women. Ethical approval was granted by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.

Cell Lines and Treatment

The HUVECs were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, Waltham, Massachusetts) at 37°C under normoxic (20% O₂) or hypoxic (1% O₂) conditions. For overexpression of WIF1, the cells were transfected with a lentivirus vector encoding the full-length human WIF1 gene (WIF1 overexpression) or empty vector (control; GenePHarma, Shanghai, China). Stable HUVECs overexpressing the WIF1 gene were established by puromycin screening before protein collection or beginning functional experiments.

Western Blotting

Proteins extracted from HUVECs were subjected to Western blotting. Briefly, cells were lysed in 200 μL of lysis buffer (Beyotime, Nanjing, Jiangsu, China). An equal amount of denatured protein per well was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to standard protocols. The separated proteins were electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts). The membranes were blotted using primary antibodies against human WIF1 (1:1000; Abcam, Cambridge, Massachusetts), β-catenin, Wnt5a (1:1000; Millipore, Darmstadt, Germany), HIF-1α, cyclin D1, VEGF, GAPDH, and β-actin (1:500; Santa Cruz, Dallas, Texas) at 4°C overnight and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000; Abcam). A densitometric analysis was performed with enhanced chemiluminescence reagents and a Chemi-doc image analyzer (Bio-Rad, Hercules, California). GAPDH and β-actin were used as a loading control. The relative density of the target protein band was normalized to the density of the GAPDH and β-actin band to represent the relative expression of the target protein.

Immunohistochemistry

Formalin-fixed, paraffin-embedded 5 μm sections were dewaxed in xylene and rehydrated through a gradient of ethanol. After quenching endogenous peroxidase activity and blocking with normal goat serum, sections were incubated with primary antibodies against human WIF1 and platelet EC adhesion molecule 1 (CD31; 1:100, Abcam) at 4°C overnight. Nonimmune rabbit or mouse immunoglobulin G was used as a negative control instead of the primary antibody. After washing in phosphate-buffered saline (PBS), the sections were incubated with biotinylated secondary antibody and stained using a diaminobenzidine kit (Zhongshan Golden Bridge Crop., Beijing, China). Sections were counterstained with hematoxylin and then observed with a microscope (Olympus LX70; Olympus, Middlesex, United Kingdom). All experiments were repeated in triplicate.

Quantitative RT-PCR (reverse transcription-polymerase chain reaction)

Total RNA extracted from HUVECs was lysed in TRIzol reagent (Invitrogen, Carlsbad, California) and reverse transcribed using a Primescript RT reagent kit (Takara Bio, Dalian, China). The primer pairs of VEGF, cyclin D1, Wnt-5a, β-catenin and HIF-1α were obtained from Takara Bio with cycling parameters as defined by the manufacturer. The relative mRNA expression was normalized to GAPDH levels and analyzed using the 2^-ΔΔCt method. The experiments were performed in triplicate.

Cell Cycle Flow Cytometric Analysis

Cells were harvested, fixed in 70% ethanol, and resuspended in 500 μL of freshly prepared propidium iodide and RNase A solution. The cell cycle distribution was quantified by investigation of the DNA content with a fluorescence-activated cell sorting Vantage SE flow cytometer (BD Biosciences, San Jose, California).

Apoptosis Flow Cytometric Analysis

Apoptotic cell death was measured using the Annexin V-PE(Phycoerythrin)/7-AAD(Aminoactinomycin D) Apoptosis Detection Kit (Key-Gen Biotech, Nanjing, China) by flow cytometry. Briefly, after treatment, cells were collected by trypsinization, washed with ice-cold PBS twice, resuspended in 50 μL binding buffer (including 5 μL of 7-AAD) for 5 minutes, then mixed with 450 μL binding buffer and stained with 1 μL of PE-conjugated Annexin V for 10 minutes. The apoptosis rate was then quantified by flow cytometry.

In Vitro Tube Formation Assay

BD Matrigel (BD Biosciences, San Jose, California) diluted with a serum-free medium at a ratio of 1:2 was distributed in a 96-well plate on ice. Pretreated HUVECs (10× 10⁴/well) were gently added to each of the triplicate wells after matrigel solidification and cultured for 6 hours. Capillary-like HUVEC structures were photographed (×200) and quantified by calculating the length of the tube-like structures with the software Image J (National Institutes of Health, Bethesda, Maryland). The tracks of ECs organizing into cellular cord (tube) networks were counted, and the results from 5 randomly selected fields were averaged. The tube formation index were calculated using normalized percentages of tube length ([mm] per mm²) to the control cells.

Wound Healing Assay

Cells were seeded in 6-well plates and were then subjected to various treatments as indicated. After reaching confluence, the cell monolayers were wounded with a sterile plastic tip and were cultured for 24 hours. The image of the wound was captured at 0 hour and 24 hours by microscopy (Nikon, Tokyo, Japan), and the wound area was assessed using Image-J software, normalized to the wound area at the 0-hour time point.

Statistical Analysis

All data were expressed as the mean ± standard deviation from 3 independent experiments. Repeated measures analysis of variance (RM ANOVA) with a Bonferroni posttest was carried out with GraphPad Prism 6.0 (GraphPad Software, San Diego, California). P < .05 was considered significantly different.

Results

1. Wnt inhibitory factor 1 was expressed in human ECs during pregnancy and was weaker in the first trimester than in the third trimester placenta

The WIF1 protein was expressed in ECs of the human placenta in different stages of pregnancy (Figure 1B and H). CD31 was used as a marker to identify ECs (Figure 1A, D and G). The expression of WIF1 protein in the ECs of the first trimester placenta (Figure 1H) was weaker than the term placenta (Figure 1B). Wnt inhibitory factor 1 was also expressed in trophoblasts of placenta (Figure 1B and H) and maternal endothelial and decidual cells (Figure 1E).

2. Wnt inhibitory factor 1 inhibited VEGF, migration, and tube formation of HUVECs

Given the weaker expression of WIF1 in the first trimester placenta, we assessed whether WIF1 had a suppressive effect on angiogenesis. We transfected HUVECs with lentivirus encoding human WIF1 gene and examined the migration or tube formation of the HUVECs. The results indicated an about 4-fold upregulation of WIF1 in HUVECs after transfection (0.50 ± 0.07 vs 2.18 ± 0.17, P < .001, Figure 2E and F). Compared to the control, the migration (P < .001, Figure 2A and C) and tube formation (P < .01, Figure 2B and D) of HUVECs were significantly inhibited by WIF1. Vascular endothelial growth factor plays a critical role in the development and maintenance of the vasculature. Vascular endothelial growth factor is transcriptionally regulated by the Wnt/β-catenin and HIF-1α pathway.²⁹ We performed Western blotting and real-time RT-PCR to investigate the regulation of VEGF by WIF1. The results indicated that WIF1 significantly decreased VEGF protein expression (P < .01, Figure 2E and G) and messenger RNA (mRNA) level (P < .01, Figure 2E and H) in HUVECs.

3. Wnt inhibitory factor 1 inhibited proliferation and induced apoptosis of HUVECs

Wnt inhibitory factor 1 is well known to impede tumorigenesis through cell cycle arrest and apoptosis induction. We investigated the cell cycle and apoptosis of HUVECs by flow cytometry. The S/G1 portion of HUVECs transfected with lentivirus encoding the WIF1 gene was decreased compared to the control (P < .05, Figure 3A and B). Cyclin D1 is a positive cell cycle regulator during the G₁/S transition and is known as a target of T cell factor (TCF)/β-catenin transcriptional complexes. The results suggested that WIF1 suppressed the mRNA and protein expression of cyclin D1 (P < .001 and P < .01, respectively, Figure 3E-G). The apoptosis rate of HUVECs was significantly increased by WIF1 (P < .05, Figure 3C and D).

4. Wnt inhibitory factor 1 inhibited β-catenin and Wnt5a in HUVECs

As an antagonist of the Wnt signal pathway, WIF1 directly interacts with Wnt ligands and abrogates their function. Wnt inhibitory factor 1 downregulation contributes to aberrant activation of Wnt/β-catenin signaling and the progression of several major human cancers.³⁰ We explored the expression of β-catenin, a hallmark of the Wnt/β-catenin signaling, in WIF1 overexpressing HUVECs. Western blotting and real-time RT-PCR showed that WIF1 significantly inhibited the mRNA and protein expression of β-catenin (P < .05 and P < .01, respectively, Figure 4A, B, and E). Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9b, and Wnt11 have been shown to interact with the immobilized WIF-domain of WIF1.³¹ We hypothesized that whether WIF1 influences the function of Wnts not only through direct interaction but also by hindering its expression. We detected the expression of Wnt5a in HUVECs and found that WIF1 also downregulated the expression of the Wnt5a mRNA and protein expression (P < .01 for both, Figure 4A, C, and F).

5. Hypoxia activated migration and tube formation of HUVECs by suppressing WIF1 and activating Wnt/β-catenin signal pathway

To further detect the regulation of the WIF1 and Wnt/β-catenin signal pathway in the hypoxic environment of the early placenta, we cultured HUVECs in 1% O₂ and investigated the influence on WIF1 and the Wnt/β-catenin signaling pathway as well as on HUVEC migration and tube formation. The wound healing assay indicated that the migration potential of HUVECs under hypoxic conditions was enhanced (P < .001, Figure 5A and C). The tube formation ability was also activated compared to the normoxic control (P < .01 for both, Figure 5B and D). Accordingly, mRNA and protein expression of VEGF (P < .01 and P < .05, respectively, Figure 5E-G) and cyclin D1 (P < .05 and P < .01, respectively, Figure 5E, H, and I) are increased under hypoxic conditions. Western blotting showed WIF1 protein was suppressed in hypoxic conditions (0.48 ± 0.03 vs 0.17 ± 0.02, P < .01, Figure 4D and G) with upregulated HIF1α mRNA and protein expression (P < .05 and P < .01, respectively, Figure 4D, L, and M). In contrast, the mRNA and protein expression of β-catenin (P < .01 for both, Figure 4D, H, and I) and Wnt5a (P < .05 and P < .01, respectively, Figure 4D, J, and K) was upregulated in hypoxia. Our results indicated that hypoxia activated migration and tube formation of HUVECs with WIF1 suppression and activation of the Wnt/β-catenin signal pathway.

Discussion

In the current study, we found that WIF1 was diversely expressed in vascular ECs of placentas at different gestational periods and was weaker in the early placenta than the term placenta; these findings provide evidence that WIF1 participates in the suppression of early placental angiogenesis. Wnt inhibitory factor 1 was also expressed in maternal endothelial and decidual cells. Decidual angiogenesis is fundamental for maternal–fetal circulation, and decidual factors influence placental angiogenesis through a paracrine mechanism. Our findings suggest that WIF1 plays a regulatory role in angiogenesis of the human placenta. We validated the antiangiogenesis role of WIF1 by inhibiting proliferation, tube formation and migration, and inducing apoptosis of ECs through antagonizing Wnt/β-catenin signaling pathway in HUVECs. We also identified that hypoxic conditions similar to the early placenta inhibited the expression of WIF1 and reversed the antiangiogenesis of WIF1 in HUVECs.

As a Wnt antagonist, WIF1 conspicuously inhibits VEGF expression and tumor angiogenesis through blocking the Wnt signaling pathway and suppressing the VEGF-induced phosphorylation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade.³² Consistently, tube formation and migration of ECs were significantly inhibited by WIF1. Our study provided several lines of evidence and demonstrated that WIF1 inhibited the functions of endothelial cells, including proliferation, migration, and tube formation. We employed a lentivirus encoding human WIF1 gene to establish stable HUVECs overexpressing the WIF1 gene. Our wound healing and tube formation assay results suggested that WIF1 inhibited the migration and angiogenesis of HUVECs. Vascular endothelial growth factor is a proangiogenic growth factor which was found decreased by WIF1 in HUVECs. Wnt inhibitory factor 1 activated the apoptosis of HUVECs, induced the accumulation of cells in G1 phase, and decreased the cells in S phase, leading to impaired cell renewal. Cyclin D1 is a positive cell cycle regulator during the G₁/S transition. It was inhibited by WIF1 in HUVECs. Our results confirmed that the antiangiogenic role of WIF1 in ECs.

Vascular endothelial growth factor and cyclin D1 are downstream targets of Wnt/β-catenin signaling pathway. We further investigated the effect of WIF1 on Wnt/β-catenin signaling in HUVECs. Our results indicated that WIF1 inhibited β-catenin and Wnt5a expression in HUVECs. Wnt5a is known as a noncanonical Wnt. It can not only activate noncanonical Wnt/Ca²⁺ signaling but also inhibit or activate canonical Wnt/β-catenin signaling. The discrepancy of functions depends on the receptor context in different cells.^33,34 Wnt5a regulates several angiogenic effectors to promote angiogenesis.³⁵ We and other researchers have found that Wnt5a expressed in placenta.³⁶ In another study, we have found that WIF1 directly interact with Wnt5a in human trophoblast cell line. So we speculate that Wnt5a participates in the angiogenesis in placenta. But no prior studies show that WIF1 inhibits Wnt ligand expression in ECs. There may be some complicated interactions including biological feedback between WIF1 and Wnt5a. Further investigations are needed to explore if the effect is direct or indirect and is there any other signaling pathway involved in it.

Current evidence supports the concept that the placenta and the fetus develop in a hypoxic environment during the first trimester.¹⁰ Hypoxia is a potent angiogenic trigger that stimulates proangiogenic factor activity.¹¹ Reports differ on the relationship between hypoxia and the Wnt/β-catenin signaling pathway. Hypoxia activates β-catenin and its downstream effectors lymphoid enhancer-binding factor 1 (LEF-1)/TCF-1 in embryonic stem cells.²⁷ However, a study in low O₂ environments of early fetal development indicated that HIF1α repressed myogenesis through inhibition of canonical Wnt signaling.³⁷ In cancer cells, HIF-1α and HIF-2α exert opposing effects on canonical Wnt signaling.³⁸ Therefore, we can speculate that the discrepancy in the hypoxic effect of Wnt signaling may be attributed to different cell types. As a modulator of Wnt signaling, the relationship between WIF1 and hypoxia in ECs and their coefficient on angiogenesis remains unclear. In this study, we found that WIF1 is inhibited under hypoxic conditions accompanied by upregulation of HIF-1α. In the previous study, WIF1 is silenced by epigenetic modification in numerous tumors.^26,27,39 The influence of hypoxic stress on gene expression through modification of DNA methylation has been confirmed in the context of tumor suppressor genes and cancer progression. We speculate that epigenetic mechanisms are important in suppressing WIF1 in response to hypoxia and increased HIF-1α. In contrast, the expression of Wnt5a and β-catenin, which has been found to be inhibited by WIF1 in this study, was increased, and target genes of Wnt signaling such as VEGF and cyclin D1 were upregulated in HUVECs. Moreover, the migration and tube formation of HUVECs were activated in hypoxia, in contrast to the effect of WIF1. These results suggest that hypoxia may activate the Wnt/β-catenin signaling pathway through suppression of WIF1. Certainly, the transcriptional activity of β-catenin needs to be further investigated. However, the relationship between HIF1α and WIF1 remains unclear. We speculate that the regulation of the Wnt/β-catenin signaling pathway may be directly by HIF1α or by HIF1α suppressed WIF1. Further investigations are needed to explore the internal interactions between HIF1α, WIF1, and the Wnt signaling pathway.

In summary, our present study supported the hypothesis that WIF1 is crucial as a negative regulator of the functions of HUVECs in angiogenesis. Hypoxia plays an important role in controlling WIF1 expression. We also demonstrated that the Wnt/β-catenin signaling pathways was activated in correspondence to the suppression of WIF1 in angiogenesis of HUVECs under hypoxic conditions. This newly identified hypoxia/WIF1 link provides a greater understanding of angiogenesis regulation under hypoxic conditions in the early placenta. In addition, although HUVECs have been used as a model for ECs in many studies that considered placental angiogenesis, nowadays, growing literature shows that the placental macrovascular ECs differ in phenotype, gene expression, and physiology from the microvascular ECs, such as those present within the placental villi known as human placental microvascular endothelial cells (HPEC).⁴⁰ Further studies using HPEC are needed to be implemented.

Footnotes

Acknowledgments

We thank for the excellent technical assistance from Key Laboratory for Major Obstetric Diseases of Guangdong Province and Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing Medical University.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work is supported by National Natural Science Foundation of China grants (No. 81070502, 81300508, 81300509 and 81471472), the general project of fundamental science and frontier technology of Chongqing Natural Science Foundation (No. cstc2015jcyjA10100), and the National Key Clinical Department Funding (grant no. 201101ckZD).

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