Original Article
The Role of Laminin a4 in Human Umbilical Vein Endothelial Cells and Pathological Mechanism of Preeclampsia
Reproductive Sciences 1-11 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115570913 rs.sagepub.com
Nan Shan, MD1, Xuemei Zhang, MD1, Xiaoqiu Xiao, PhD2, Hua Zhang, MD1, Ying Chen, MD1, Xin Luo, MD1, Xiru Liu, MD1, Baimei Zhuang, MD1, Wei Peng, MD1, and Hongbo Qi, MD1
Abstract Preeclampsia (PE) is associated with defective placental angiogenesis and poor placentation. Laminins are the main noncollagenous glycoproteins in basement membranes, and laminin a4 (LAMA4) promotes the migration, proliferation, and survival of various cells. The primary purpose of this study is to investigate the role of LAMA4 in human umbilical vein endothelial cells (HUVECs) function during the development of PE. We found expression levels of LAMA4 in human PE placentas were significantly lower compared to the control placentas. The LAMA4 small-interfering RNA transfection and hypoxia–reoxygenation (H/R) intervention reduced the migratory and tube formation abilities of HUVECs. The mitogen-activated protein kinase (MAPK) signaling pathways interacted with LAMA4 expression and H/Rexposure led to MAPK pathways activation in HUVECs. We demonstrated that LAMA4 is very crucial in promoting the functions of endothelial cells. Oxidative stress plays a vital role in controlling expression of LAMA4 through MAPK signaling pathways, which suggests a possible pathological mechanism of PE. Keywords oxidative stress, laminin a4, p38 mitogen-activated protein kinase, preeclampsia
Introduction Preeclampsia (PE) is a pregnancy-specific obstetric complication characterized not only by hypertension but also by a high level of proteinuria after 20 weeks of gestation.1 It affects 5% to 7% of all healthy pregnancies and causes 70 000 to 80 000 maternal deaths and 500 000 perinatal deaths worldwide annually.2 The pathological mechanism of PE is still unclear, but the development of this pregnancy disorder may be mainly induced by excessive oxidative stress or abnormal shallow vascular endothelial growth. Angiogenesis is essential for the establishment and execution of the bidirectional maternal–fetal exchange of nutrients and respiratory gases, which is arguably the most important event for pregnancy.3 There is strong evidence that defective placental angiogenesis contributes to poor placentation and is essential for the development of PE.4 The blood vasculature consists of different types of vessels with various cellular and extracellular matrix (ECM) compositions that can impact the integrity of vascular structure and function. These compositions include proteoglycans and glycoproteins such as fibronectin, laminin, entactin, tenascin, and thrombospondin. Laminins are a family of at least 15 heterotrimeric, cruciform, or T-shaped glycoproteins, each formed by 1 of the
5 a, 3 b, and 3 g chains5. Laminins are the first and major basement membrane components appearing during the early stages of embryonic development and display a remarkable repertoire of biological functions.6,7 Laminin a4 (LAMA4), a component of Lm-411 and Lm-421, has been found in various tissues of mesenchymal origin, endothelial, and some epithelial basement membranes (BMs).8-10 Numerous analyses indicate that the subunit LAMA4 will dramatically impact the behaviors of endothelial cells via complex interaction with integrin receptors on the surface of endothelial cells.11 However, the role of LAMA4 in the placenta is largely unknown. Therefore, we aimed to explore the effect of LAMA4 during the whole pregnancy and its potential function during development of PE.
1 Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China 2 Laboratory of Lipid & Glucose Research, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
Corresponding Author: Hongbo Qi, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China. Email: [email protected]
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Oxidative stress has been implicated in the pathology of many hypertensive pregnancy-related conditions, including PE and gestational hypertension.12 Reactive oxygen species (ROS) as molecules produced from the reduction in molecular oxygen are the by-products of aerobic respiration and metabolism. These molecules with diverse chemical properties are capable of activating various signaling pathways.13 For instance, the stress-responsive p38 mitogen-activated protein kinase (MAPK) pathway is reported to be robustly activated in PE and is essential to oxidative stress-mediated placental dysfunction.14 It is hypothesized that intermittent placental perfusion, secondary to the deficient trophoblast invasion of endometrial arteries, will induce an ischemia–reperfusion (I/R) insult to the placenta and cause intense oxidative stress in the placental endothelium.15 The disruption of endothelial homeostasis is fundamental to the initiation and progression of PE.16 Mitogen-activated protein kinases cascades transduce a diverse spectrum of extracellular and intracellular stimuli into alterations in gene expression and cellular function. Reports from Reich et al have showed that MAPK are involved in laminin signaling.17 However, few data are available on the potential function of LAMA4 in the process of placental oxidative stress and MAPK signaling pathway. So, we mimicked the pathological condition of PE using a hypoxia–reoxygenation (H/R) circle and utilized RNA interference (RNAi) transfection to further evaluate the effects of LAMA4 on endothelial cell migration and tube formation in vitro. In addition, the p38 MAPK pathway inhibitor SB203580 was applied to study the possible molecular and cellular mechanisms of the LAMA4-p38 MAPK signaling pathway in human umbilical vein endothelial cells (HUVECs). The above-mentioned evidences support the function of LAMA4 in promoting angiogenesis of HUVECs and also suggest that the dysregulation of LAMA4 expression may be associated with the occurrence of PE.
Human placental tissues from 6 to 8 weeks (n ¼ 9) were obtained from healthy women undergoing legal abortion for nonmedical reasons; normal term placentas (n ¼ 15) and PE placentas (n ¼ 17) were collected after cesarean operations. These tissues were collected and washed in 0.9% saline, and then 1 piece of the tissues was fixed in 3% formaldehyde and embedded with paraffin at room temperature for immunohistochemistry (IHC).
Cell Culture and H/R Application The HUVEC cell line was used in our study. The HUVECs were cultured in Dulbecco Modified Eagle Media: Nutrient Mixture F12 (DMEM F12; Gibco, Massachusetts) containing 10% fetal bovine serum (FBS; Gibco). The cells were maintained under standard culture conditions at 37 C under 5%CO2. Then H/R intervention (8 hours under 2% oxygen and 16 hours under standard culture conditions, 2 cycles) was performed in a trigas cell culture incubator at 37 C.
RNA interference and p38 MAPK Inhibition The cells for RNAi were transfected with 100 nmol/L LAMA4 small-interfering RNA (siRNA; 5’-CAGGGAUUUAUGCAGAAAUTT-3’, GenePharma, Shanghai, China; Genbank ID for LAMA4: NM_000006.12) or the control siRNA (a universal negative control, GenePharma) with Lipofectamine 2000 (Invitrogen, Carlsbad, California) as recommended by the manufacturer. Transfection was performed according to the manufacturer’s protocol. The transfection efficiency measured using fluorescence-labeled siRNA was >90%. In order to block the p38 MAPK pathway, SB203580 (Invitrogen, Carlsbad, Calif., USA) was dissolved in dimethyl sulfoxide (DMSO) to 5 mmol/L and then used to preincubate the cells as described previously.18
Immunohistochemistry Method and Material Patients and Tissue Collection The definition and criteria of PE were based on the diagnostic criteria outlined by the American College of Obstetrics and Gynecology. Preeclampsia was characterized by systolic blood pressure 140 mm Hg or diastolic blood pressure 90 mm Hg, together with proteinuria (0.3 g protein in a 24-hour urine collection, usually corresponding to 1 þ on urine dipstick tests) that occurred after 20 weeks of gestation in previously normotensive women. Patients with diabetes mellitus, chronic renal diseases, chronic hypertension, or other metabolic diseases were excluded from this study. This study and the use of samples were conducted under standard experimental protocols approved by the Ethics Committee of Chongqing Medical University. Ethical approval was granted by the Ethics Committee of the First Hospital of Chongqing University. All women were informed and signed consent to donate their placentas for scientific research use. 2
Immunohistochemistry was performed as described previously.19 The sections (each 5 mm thick) were dewaxed, rehydrated, and incubated with 3% H2O2 for 10 minutes to quench the endogenous peroxidase. After blocking with 20% normal goat serum for 30 minutes, the sections were incubated with a primary LAMA4 antibody (1:500) and a platelet endothelial cell adhesion molecule 1 (CD31) antibody (1:500) overnight at 4 C. After washing with phosphate buffer solution (PBS), the sections were incubated with biotinylated secondary antibody and stained using diaminobenzidine solution. Nonimmune rabbit immunoglobulin G was used as a negative control. Each experiment was performed at least three times, and comparable results were obtained each time.
Western Blotting The control and treated cells were homogenized in a lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China), cracked by an ultrasonic probe for 2 minutes and then
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centrifuged at 14 000g at 4 C for 15 minutes to remove the debris. Protein contents were measured using a bicinchoninic acid (BCA) Protein Assay (Beyotime). 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 electro-transferred to polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts). After blockage, the membranes were incubated with appropriate primary antibodies overnight at 4 C, including rabbit antibodies against LAMA4 (1:1000; Sigma-Aldrich Co, St. Louis, Missouri), p38, p-p38, c-Jun NH2-terminal protein kinase (JNK), p-JNK, extracellular signal-regulated kinase (ERK), p-ERK (all 1:1,000; Cell Signaling Technology, Danvers, Massachusetts), and b-actin (1:1000; Santa Cruz Biotechnology, Dallas, Texas). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) at 37 C for 2 hours. Positive bands were detected by enhanced chemiluminescence reagents (Beyotime) and analyzed by Quantity One 4.4.0 (Bio-Rad Laboratories, Hercules, California). Experiments were performed in triplicate.
RNA Extraction and Quantitative Real-Time ReverseTranscriptase Polymerase Chain Reaction Total RNA was extracted from the control and treated cells using a TRIzol regent (Takara Biotechnology, Japan) and purified according to the manufacturer’s instruction. Reverse transcription was performed using a Primescript RT reagent kit (TaKaRa). Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) was performed on a C1000 Thermal Cycler (Bio-Rad). The means of threshold cycles were normalized to atubulin levels, and the relative messenger RNA (mRNA) levels were analyzed using the 2DDCt method. The experiments were performed in triplicate. The primers used in this study include LAMA4 (Forward: 5’-GGAAAATAAGCGAGGCACCG’, Reverse: 5’-AGCCACAGAGGCAGAACCGA-3’) and atubulin (Forward: 5’-CCAA GTTCTGGGAGGTGATCTG 3’, Reverse: 5’- TTGTAGTAGACGTTGATGCGCTC-3’).
Migration Assay The migration assay was performed in 24-well plates (Costar, Cambridge, Massachusetts) and polycarbonate filters (8-mm pores; Transwell Chamber, Millipore) as described previously.20 The HUVECs (1.0 105/well) with or without treatments (including siRNA and SB) were seeded into the upper chamber in a serum-free medium, whereas a medium containing 10% FBS was added to the lower chamber under normoxia at 37 C for 48 hours. The H/R group was differently treated: Normal cells were exposed to H/R under 37 C for 48 hours. Then, the inserts were fixed with methyl alcohol and stained with 3% Crystal violet. The migratory cells were observed and recorded under a microscope (Olympus IX51, Japan) at a magnification of 200 in 10 random fields. The relative migration percentages were calculated with nontreated cells cultured
under normal conditions with an average migration percentage of 100%. All experiments were repeated triple times.
Tube Formation BD Matrigel (cat. no. 356230, BD Biosciences, San Jose, California) was diluted with a serum-free medium at a ratio of 1:2 with cooled pipettes and distributed in a 24-well plate (150 mL/well) on ice. As before, HUVECs (1.0 105/well) with various pretreatments were gently added to each of the triplicate wells after matrigel solidification, followed by H/R intervention or normal culture for 48 hours. Digital images were captured from each well (200). Tube formation was quantified by calculating the total tube length of tube-like structures using a camera (Olympus) interfaced with the software ImageJ (National Institutes of Health, Bethesda, Maryland). The tracks of endothelial cells organizing into cellular cord (tube) networks were counted, and the results from 5 randomly selected fields were averaged. The normalized percentages were calculated using nontreated cells cultured under normal conditions with an average tube formation index [tube length (mm) per mm2] of 100%.
Cell Proliferation Assay After transfection of siRNA, the HUVECs were subjected into 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazo-lium bromide (MTT) assay to test cell proliferation. The HUVECs (5 103/well) were seeded in 96-well plates. After a 20-hour culture, 100 mL of an MTT reagent was added to the medium and gently removed 4 hours later. The DMSO (100 ml) was then added into each well, and the optical density of each well was assessed at wave length 570 nm. Each experiment was performed in triplicate.
Flow Cytometry Analysis of Cell Apoptosis The cell apoptosis rate was measured by an annexin V-fluorescein isothiocyanate (FITC) and PI apoptosis detection kit (Key-GenBiotech, JiangSu, China) as described previously.21 After culture for 48 hours, the cells after various pretreatments were stained with a binding buffer (1 mg/mL propidium iodide and 1 mg/mL FITC-labeled annexin-V) and gently vortexed, then incubated in the dark—at room temperature for 30 minutes. Apoptosis rates were quantified with an FACS Vantage SE Flow Cytometry (FCM) meter (BD Biosciences). The cells that excluded PI and were positive for Annexin V-FITC binding were represented as apoptotic cells.
Immunofluorescence Staining The normal HUVECs and the H/R-intervened cells were permeabilized with 0.2% Triton X-100 and blocked with PBS containing 1% BSA for 1 hour after fixation in ice methanol. An anti-LAMA4 antibody (1:100) was used as the primary antibody. After washing with PBS, the cells were incubated with an FITC secondary antibody (Zhongsan Golden Bridge
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Figure 1. LAMA4 was highly expressed in human endothelial cells during pregnancy. A, Immunostaining of LAMA4 in human villi; its protein was intensely and specifically expressed in syncytotrophoblasts and endothelial cells in the first trimester. B, Expression of CD31 in human villi in the first trimester, it was as a marker of endothelial cells. C, Negative control with nonimmune rabbit IgG in human villi. D, LAMA4 was strongly expressed in human decidua in the first trimester, especially in the endothelial cells and decidual cells. E, Expression of CD31 in human decidua. F, Negative control with nonimmune rabbit IgG in the maternal decidua. G, Expression of LAMA4 was also found in endothelial cells of the normal term placenta. H, LAMA4 was rarely expressed in the placenta of preeclampsia. I, Negative control with nonimmune rabbit IgG in the term placenta. Scale bar ¼ 100 mm. LAMA4 indicates laminin a4; IgG, immunoglobulin.
Crop, Beijing, China) for 1 hour. The nuclei were stained with 3 mg/mL propidium iodide for 8 to 10 minutes. Images were acquired with an Olympus BMX-60 microscope equipped with a cooled charge-coupled device sensi-camera (Cooke, Auburn Hills, Michigan) and the software Slidebook (Intelligent Imaging Innovations, Denver, Colorado). All experiments were repeated in triplicate.
Statistical Analysis Each experiment was performed in triplicate. All the values are expressed as mean + standard error of the mean. The data were analyzed for significance using the software GraphPad Prism (GraphPad Software, San Diego, California). Between the 2 groups, comparisons of continuous variables were conducted using independent t tests. Statistical differences among multiple groups were evaluated by 1-way analysis of 4
variance (ANOVA), followed by the least significant difference multiple comparison as appropriate. P < .05 was considered as significant.
Results 1.
LAMA4 was highly expressed in human endothelial cells during the first trimester.
The LAMA4 protein expressions in human placental villi at different stages of pregnancy were first evaluated using IHC. CD31 was as a marker used to identify endothelial cells (Figures 1B & 1E). During the first trimester, LAMA4 protein was intensely and specifically expressed in syncytiotrophoblasts and endothelial cells (Figures 1A & 1D). Especially, in maternal decidua, LAMA4 was strongly stained in endothelial cells and decidual cells (Figure 1D). In the third trimester,
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Figure 2. Effects of H/R, siRNA, and p38 inhibitor SB203580 on expressions of LAMA4, VEGFR2, and MAPK signaling pathways in HUVECs. A, Protein expressions of LAMA4, VEGFR2, p-p38, and T-p38 (total p38) were examined by Western blotting. B, Statistical analysis of the Western blotting results in (A; n ¼ 3 in triplicate, *P < .05 and **P < .01compare with the control group). C, Representative Western blots of ERK, JNK, p-ERK, and p-JNK proteins in HUVECs with indicated treatments. D, Statistical analysis of the Western blotting results in (C; n ¼ 3 in triplicate, **P < .01compared with the control group). Data were analyzed by ANOVA to assess significant differences. H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; VEGFR2, Vascular endothelial growth factor receptor 2; MAPK, mitogen-activated protein kinase; HUVECs, human umbilical vein endothelial cells; ERK, extracellular signal-regulated kinase; JNK, NH2-terminal protein kinase; p-ERK phophorylated ERK; p-JNK, phophorylated JNK; ANOVA, analysis of variance.
Figure 3. Effects of H/R and siRNA on LAMA4 mRNA expression in HUVECs. A, mRNA expression of LAMA4 was decreased by siRNA transfection examined by RT-qPCR (n ¼ 3 in triplicate, P < .01). B, H/R treatment deduced LAMA4 mRNA expression by almost 60% (n ¼ 3 in triplicate, P < .01). Data were analyzed by t test to assess significant differences. H/R indicates hypoxia–reoxygenation; siRNA, smallinterfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; mRNA, messenger RNA; RT-qPCR, quantitative realtime reverse-transcriptase polymerase chain reaction.
LAMA4 was still expressed in endothelial cells of the normal placentas (Figure 1G) but was even less expressed in PE placentas (Figure 1H). 2.
The LAMA4 siRNA significantly inhibited migration and tube formation of HUVECs.
Given the evident expression of LAMA4 in the firsttrimester placentas, we next investigated whether LAMA4 had
effect on the functions of endothelial cells. We examined the migration or tube formation of HUVECs using siRNA transfection tests. The Western blotting and RT-qPCR showed that siRNA targeting significantly decreased the LAMA4 protein expression in HUVECs (Figures 2A and 3A). Vascular endothelial growth factor receptor 2 (VEGFR2) is expressed most prominently in vascular endothelial cells and their embryonic precursors. This receptor has potent tyrosine kinase activity and can transduce the major signals for
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Figure 4. Migratory ability of HUVECs decreased under the H/R circumstance and LAMA4 downregulation. Representative images showed HUVECs passing through the membrane by migration assay (magnification 200). A-D, Representative images showed HUVECs penetration through the membrane. A, Nontreated cells cultured under normal condition; (B) siRNA group; (C) SB group in which cells were preincubated with SB203580 and cultured under normoxic conditions; (D) H/R group in which cells were maintained under hypoxic conditions for 8 hours followed by reoxygenation for 16 hours, 2 circles. E, Bar graph showed relative numbers of migrating cells (%). Data were analyzed by ANOVA to assess significant differences (n ¼ 3 in triplicate, **P < .01 compared with the control group). H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; ANOVA, analysis of variance; SB, SB203580 4(4– Fluorophenyl)–2–(4–methylsulfinylphenyl)–5–(4–pyridyl) 1 H–imidazole.
angiogenesis.22,23 We tried to investigate the relation between LAMA4 and VEGFR2 expressions and found that the expression levels of VEGFR2 decreased significantly by 1.53-fold after LAMA4 down-regulation (n ¼ 3, P < .001; Figure 2A). Cells after transfection were plated onto a pure filter for determination of migration. Compared with the control group, the LAMA4 siRNA treatment significantly decreased 6
the percentage of cells that migrated to the other side of the filter (n ¼ 3, P < .001, Figures 4A, B, and E). We also examined whether LAMA4 influenced the tube formation in HUVECs. It was noticed that LAMA4 siRNA transfection for 48 hours reduced the tube formation capability of HUVECs (n ¼ 3, p < 0.001, Figures 5A, 5C, & 5E) but not proliferation and/or apoptosis (n ¼ 3, p > 0.05, Figures 6A, B, E, and F).
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Figure 5. Tube formation ability of HUVECs decreased under the H/R circumstance and LAMA4 siRNA transfection. A-D, Representative images revealed tube formation in HUVECs. A, Nontreated group; (B) H/R group; (C) siRNA group; (D) SB group. E, Bar graph showed the normalized tube formation index to the control group in the tube formation assay (%).The tube formation index was expressed as tube length (mm) per area (mm2). Bars ¼ 100 mm. Data were analyzed by ANOVA to assess significant differences (n ¼ 3 in triplicate, **P < .01 compared with the control group). H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; ANOVA, analysis of variance.
3.
The H/R condition suppressed migration and tube formation of HUVECs.
To further detect the potential function of LAMA4 in PE, we used H/R condition to mimic the oxidative stress environment during PE development. Western blotting showed that LAMA4
protein expression was down-regulated by about 92.48% in the HUVECs under H/R treatment compared with the control group (n ¼ 3, P < .001; Figures 2A and 2B). RT-qPCR also consistently showed that LAMA4 mRNA level was reduced after H/R exposure (n ¼ 3, P < .01; Figure 3B). Immunofluorescence also testified that H/R treatment significantly decreased
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Figure 6. The effects of different treatments on apoptosis rates in HUVECs, assessed by flow cytometry. A-D, Representative pictures of flow cytometry for apoptosis rate among normal nontreated HUVECs and different treatments, including H/R, siRNA, and SB. E, MTT assay showed no significant difference in cell proliferation using LAMA4 siRNA treatment in HUVECs. F, LAMA4 siRNA transfection and SB had no significant effects on the apoptosis rate in HUVECs by FCM, but H/R exposure significantly increased the apoptosis rate in HUVECs. Data were analyzed by ANOVA to assess significant differences (n ¼ 3 in triplicate, **P < .01 comparing with the control group). H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; ANOVA, analysis of variance; FCM, flow cytometry; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazo-lium bromide.
the LAMA4 protein expression in HUVECs (Figure 7). Interestingly, consistent with the decline in LAMA4 expression, the VEGFR2 expression level also decreased dramatically after exposure to H/R for 48 hours, tested by Western blotting (n ¼ 3, P < .001, Figure 2A). In addition, our results demonstrated that the migration potential of H/R-treated HUVECs diminished to 50.21% compared with the nontreated cells (n ¼ 3, P < .001, Figures 4A, D, and E). The H/R intervention also reduced the tube formation potential of HUVECs by 43.93% compared to the control group (n ¼ 3, P < .001, Figure 5A, 5B, and 5E). Furthermore, FCM was used to quantify the effect of H/R on apoptosis in HUVECs. A representative FCM image in Figure 6 shows that the H/R environment dramatically promoted the cell apoptosis (n ¼ 3, P < .001; Figures 6A, 6C, and F). 4.
Interactions between MAPK signaling pathway and LAMA4 expression in HUVECs.
The classic oxidative stress-p38 MAPK signaling pathway was selected to explore the mechanism of LAMA4 in 8
HUVECs. Compared with the control group, the p38 inhibitor SB203580 markedly reduced the LAMA4 expression in HUVECs, which was consistent with the reduction in VEGFR2 expression (both n ¼ 3, P < 0.001, Figures 2A & 2B). Moreover, H/R treatment induced significant up-regulation of phosphorylation of p38 (or p-p38) in HUVECs. The bar graphs presenting the quantification of proteins revealed a nearly 2-fold higher level of p-p38 in the H/R group compared with the control group (Figures 2A and B). Since the family of MAPK protein kinases controls many cellular events such as cell proliferation, differentiation, apoptosis, embryogenesis, inflammation, and stress responses,24 we also investigated the expressions of other MAPK proteins after LAMA4 siRNA treatment and H/R exposure, including JNK and ERK. In Figure 2C and D, the results showed that H/R treatment induced higher level of phosphorylation of ERK (p-ERK; n ¼ 3, P < .001) but not phosphorylation of JNK (p-JNK; n ¼ 3, P > .05). However, down-regulation of LAMA4 expression decreased the level of p-JNK (n ¼ 3, P < .01) but not p-ERK (n ¼ 3, P > .05). Our data indicated that although MAPK pathways interacted with LAMAA4, but p38, ERK, and JNK played different roles in regulating
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Figure 7. Immunofluorescent detections of LAMA4 proteins (green) and nuclei (red, propidium iodide, PI) in nontreated and H/R HUVECs. LAMA4 (green) expression decreased in HUVECs after H/R treatment for 48 hours (n ¼ 3 in triplicate). All the pictures were captured 400. /R indicates hypoxia–reoxygenation; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells. (The color version of this figure is available in the online version at http://rs.sagepub.com/).
expression of LAMA4 in endothelial cells and responding to oxidative stress.
Discussion The underlying pathophysiology of PE involves the dysregulations of endothelial function in both the maternal and the fetoplacental circulations. Oxidative stress resulting from overproduction of ROS impairs development of the placental vasculature and has been proposed as a key intermediary step during the pathogenesis of PE. The LAMA4 is ubiquitously localized in endothelial BMs throughout various vessels and independent of the developmental stage.25 The LAMA4 is expressed in the mouse developing endothelium from early stages of embryonic development (E8.5).26 In vitro assays suggest several potential roles of LAMA4 in angiogenesis, including cell attachment, cell migration, and tubule formation.27 Our study provided several lines of evidence and demonstrated that LAMA4 promoted the functions of endothelial cells, including migration and angiogenesis. Meanwhile, H/R condition was used to elucidate the underlying oxidative stress-mediated signaling pathways between the effect of LAMA4 and the development of PE. Laminins have been found in the pericellular basement membranes of human decidual cells and placental villi.28 In
this study, LAMA4 was strongly expressed in endothelial cells in placental villi and decidua. The LAMA4 was also highly expressed in the third-trimester placentas compared to the PE placentas. During early pregnancy, decidual angiogenesis forms a new vascular network that serves as the first exchange apparatus between maternal circulation and the developing embryo. Thus, decidual angiogenesis is a fundamental process deciding embryonic survival and successful pregnancy. Our findings suggested that LAMA4 played a role in regulating angiogenesis and placentation in human placentas. Vascular endothelial growth factor plays a key role in decidual angiogenesis and function.29 To further verify the regulatory role of LAMA4 on HUVECs, we investigated the expression of VEGFR2, which was found consistent with the reduction of LAMA4 by siRNA transfection. Downregulation of LAMA4 also decreased cell migration and tube formation potentials in HUVECs. This study proved that LAMA4 may be directly involved in the migration and angiogenesis functions of endothelial cells. To test the effects of long-term H/R on HUVECs and LAMA4, we prolonged the hypoxia to 8 hours and the reoxygenation to 16 hours and extended to 2 circles. The migration and tube formation potentials of HUVECs were dramatically weakened after H/R treatment, accompanied with reduced expressions of LAMA4 and VEGFR2. The above-mentioned evidence strongly suggested that dysregulation of LAMA4 was
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associated with the impaired angiogenesis function induced by oxidative stress. A growing number of studies show that MAPKs are involved in laminin signaling.17 The MAPK pathways can be activated by a wide variety of different stimuli acting through diverse receptor families and regulated by phosphorylation. The 3 major mammalian MAPK subgroups include p38 MAPK, ERKs, and JNKs/ stress-activated protein kinase (SAPKs).30 The ERK pathway is mainly responsive to mitogens and growth factors and capable of modulating cell survival, migration, and apoptosis.31 The JNK and p38 pathways are activated in response to chemical and environmental stress and to inflammatory cytokines.30 We proved that inhibition of p38 pathway abates the expressions of LAMA4 and VEGFR2 in HUVECs. In addition, p-p38 was expressed at higher level after the H/R treatment, accompanied with the downregulation of LAMA4 expression. Our findings indicated that oxidative stress could lead to p38 activation and abates the impact of LAMA4 in HUVECs. Furthermore, we noticed that p-JNK was reduced by the LAMA4 siRNA transfection, and its activation in HUVECs could be restored by H/R. However, p-ERK was not affected by LAMA4 dysregulation but by H/R exposure. Taken together, our study demonstrated that p38 and JNK pathways directly interacted with LAMA4 expression in endothelial cells, and activation of p38 and ERK pathways induced by oxidative stress were related to down-regulation of LAMA4. These findings indicated that the LAMA4-MAPK pathway is likely to be responsible for the poorly developed fetoplacental vasculature associated with PE. Without doubt, LAMA4 might be regulated by different pathways under the various conditions in endothelial cells, which needs further explorations. The biological effects of laminin are mediated by receptors that are divided into 2 major groups, integrin and nonintegrin receptors. Coregulation and physical association between the 2 receptors are involved in regulating or stabilizing the interaction of laminin.32 For an instance, the interaction of the a6b1 integrin with laminin-8 (LM-411) might promote endothelial cell migration in vivo, since their expression patterns overlap with those of newly formed capillaries, where endothelial cells are actively migrating.33 Hence, further investigations need to carry on exploring the internal interactions between these receptors signaling transductions and LAMA4 function. In summary, our present study supported that LAMA4 is very crucial in promoting the functions of endothelial cells. Oxidative stress plays an important role in controlling the LAMA4 expression. We also demonstrated that MAPK signaling pathways interact with LAMA4 expression in endothelial cells under oxidative stress. These findings provide a new aspect about the cause of PE. Our study represented a good starting point for subsequent studies to further verify the role of LAMA4 in differentiation/ invasion of trophoblasts. Nevertheless, more elaborate models, such as placental explants and PE animal models are needed to more extensively study the role of the LAMA4-MAPK signaling pathways in the development of PE. Further investigations of LAMA4 will provide insight into the establishment of novel diagnostic, therapeutic, and preventative strategies for PE. 10
Acknowledgments We thank Dr Yubin Ding for helping design the article and suggestions. Authors are grateful 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) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article:: This work is supported by National Natural Science Foundation of China grants (nos. 81070502, 81170585, 81100444, 81300509, and 81300508), and the funding of National Key Clinical Department in China.
References 1. Coolman M, de Groot CJ, Jaddoe VW, Hofman A, Raat H, Steegers EA. Medical record validation of maternally reported history of preeclampsia. J Clin Epidemiol. 2010;63(8): 932-937. 2. Hutcheon JA, Lisonkova S, Joseph KS. Epidemiology of preeclampsia and the other hypertensive disorders of pregnancy. Best Pract Res Clin Obstet Gynaecol. 2011;25(4):391-403. 3. Reynolds LP, Redmer DA. Angiogenesis in the placenta. Biol Reprod. 2001;64(4):1033-1040. 4. Lash GE, Naruse K, Innes BA, Robson SC, Searle RF, Bulmer JN. Secretion of angiogenic growth factors by villous cytotrophoblast and extravillous trophoblast in early human pregnancy. Placenta. 2010;31(6):545-548. 5. Timpl R, Brown JC. The laminins. Matrix Biol. 1994;14(4): 275-281. 6. Ekblom P, Lonai P, Talts JF. Expression and biological role of laminin-1. Matrix Biol. 2003;22(1):35-47. 7. Ryan MC, Christiano AM, Engvall E, et al. The functions of laminins: lessons from in vivo studies. Matrix Biol. 1996; 15(6):369-381. 8. Iivanainen A, Sainio K, Sariola H, Tryggvason K. Primary structure and expression of a novel human laminin alpha 4 chain. FEBS Lett. 1995;365(2-3):183-188. 9. Lefebvre O, Sorokin L, Kedinger M, Simon-Assmann P. Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine. Dev Biol. 1999; 210(1):135-150. 10. Petajaniemi N, Korhonen M, Kortesmaa J, et al. Localization of laminin alpha4-chain in developing and adult human tissues. J Histochem Cytochem. 2002;50(8):1113-1130. 11. Gonzalez AM, Gonzales M, Herron GS, et al. Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo. Proc Natl Acad Sci U S A. 2002;99(25):16075-16080. 12. Burton GJ, Jauniaux E. Oxidative stress. Best Pract Res Clin Obstet Gynaecol. 2011;25(3):287-299.
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13. Paravicini TM, Touyz RM. Redox signaling in hypertension. Cardiovasc Res. 2006;71(2):247-258. 14. Shin JK, Jeong YT, Jo HC, et al. Increased interaction between heat shock protein 27 and mitogen-activated protein kinase (p38 and extracellular signal-regulated kinase) in pre-eclamptic placentas. J Obstet Gynaecol Res. 2009;35(5):888-894. 15. Hung TH, Skepper JN, Burton GJ. In vitro ischemiareperfusion injury in term human placenta as a model for oxidative stress in pathological pregnancies. Am J Pathol. 2001; 159(3):1031-1043. 16. Cudmore M, Ahmad S, Al-Ani B, et al. Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase1. Circulation. 2007;115(13):1789-1797. 17. Givant-Horwitz V, Davidson B, Reich R. Laminin-induced signaling in tumor cells: the role of the M(r) 67,000 laminin receptor. Cancer Res. 2004;64(10):3572-3579. 18. Xiong Y, Liebermann DA, Tront JS, et al. Gadd45a stress signaling regulates sFlt-1 expression in preeclampsia. J Cell Physiol. 2009;220(3):632-639. 19. Yang Z, Bai B, Luo X, et al. Downregulated Kruppel-like factor 8 is involved in decreased trophoblast invasion under hypoxiareoxygenation conditions. Reprod Sci. 2014;21(1):72-81. 20. Marconcini L, Marchio S, Morbidelli L, et al. c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro. Proc Natl Acad Sci U S A. 1999; 96(17):9671-9676. 21. Luo X, Yao ZW, Qi HB, et al. Gadd45alpha as an upstream signaling molecule of p38 MAPK triggers oxidative stress-induced sFlt-1 and sEng upregulation in preeclampsia. Cell Tissue Res. 2011;344(3):551-565. 22. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006;7(5):359-371. 23. Koch S, Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors. Cold Spring Harb Perspect Med. 2012;2(7):a006502.
24. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 2001;81(2):807-869. 25. Sorokin LM, Pausch F, Frieser M, Kroger S, Ohage E, Deutzmann R. Developmental regulation of the laminin alpha5 chain suggests a role in epithelial and endothelial cell maturation. Dev Biol. 1997;189(2):285-300. 26. Frieser M, Nockel H, Pausch F, et al. Cloning of the mouse laminin alpha 4 cDNA. Expression in a subset of endothelium. Eur J Biochem. 1997;246(3):727-735. 27. Li J, Zhou L, Tran HT, et al. Overexpression of laminin-8 in human dermal microvascular endothelial cells promotes angiogenesis-related functions. J Invest Dermatol. 2006;126(2): 432-440. 28. Wewer UM, Faber M, Liotta LA, Albrechtsen R. Immunochemical and ultrastructural assessment of the nature of the pericellular basement membrane of human decidual cells. Lab Invest. 1985; 53(6):624-633. 29. Douglas NC, Tang H, Gomez R, et al. Vascular endothelial growth factor receptor 2 (VEGFR-2) functions to promote uterine decidual angiogenesis during early pregnancy in the mouse. Endocrinology. 2009;150(8):3845-3854. 30. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 2002;298(5600):1911-1912. 31. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res. 2000;87(6):434-439. 32. Ardini E, Tagliabue E, Magnifico A, et al. Co-regulation and physical association of the 67-kDa monomeric laminin receptor and the alpha6beta4 integrin. J Biol Chem. 1997;272(4): 2342-2345. 33. Sixt M, Engelhardt B, Pausch F, Hallmann R, Wendler O, Sorokin LM. Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis. J Cell Biol. 2001;153(5):933-946.
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The Role of Laminin a4 in Human Umbilical Vein Endothelial Cells and Pathological Mechanism of Preeclampsia
Reproductive Sciences 1-11 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115570913 rs.sagepub.com
Nan Shan, MD1, Xuemei Zhang, MD1, Xiaoqiu Xiao, PhD2, Hua Zhang, MD1, Ying Chen, MD1, Xin Luo, MD1, Xiru Liu, MD1, Baimei Zhuang, MD1, Wei Peng, MD1, and Hongbo Qi, MD1
Abstract Preeclampsia (PE) is associated with defective placental angiogenesis and poor placentation. Laminins are the main noncollagenous glycoproteins in basement membranes, and laminin a4 (LAMA4) promotes the migration, proliferation, and survival of various cells. The primary purpose of this study is to investigate the role of LAMA4 in human umbilical vein endothelial cells (HUVECs) function during the development of PE. We found expression levels of LAMA4 in human PE placentas were significantly lower compared to the control placentas. The LAMA4 small-interfering RNA transfection and hypoxia–reoxygenation (H/R) intervention reduced the migratory and tube formation abilities of HUVECs. The mitogen-activated protein kinase (MAPK) signaling pathways interacted with LAMA4 expression and H/Rexposure led to MAPK pathways activation in HUVECs. We demonstrated that LAMA4 is very crucial in promoting the functions of endothelial cells. Oxidative stress plays a vital role in controlling expression of LAMA4 through MAPK signaling pathways, which suggests a possible pathological mechanism of PE. Keywords oxidative stress, laminin a4, p38 mitogen-activated protein kinase, preeclampsia
Introduction Preeclampsia (PE) is a pregnancy-specific obstetric complication characterized not only by hypertension but also by a high level of proteinuria after 20 weeks of gestation.1 It affects 5% to 7% of all healthy pregnancies and causes 70 000 to 80 000 maternal deaths and 500 000 perinatal deaths worldwide annually.2 The pathological mechanism of PE is still unclear, but the development of this pregnancy disorder may be mainly induced by excessive oxidative stress or abnormal shallow vascular endothelial growth. Angiogenesis is essential for the establishment and execution of the bidirectional maternal–fetal exchange of nutrients and respiratory gases, which is arguably the most important event for pregnancy.3 There is strong evidence that defective placental angiogenesis contributes to poor placentation and is essential for the development of PE.4 The blood vasculature consists of different types of vessels with various cellular and extracellular matrix (ECM) compositions that can impact the integrity of vascular structure and function. These compositions include proteoglycans and glycoproteins such as fibronectin, laminin, entactin, tenascin, and thrombospondin. Laminins are a family of at least 15 heterotrimeric, cruciform, or T-shaped glycoproteins, each formed by 1 of the
5 a, 3 b, and 3 g chains5. Laminins are the first and major basement membrane components appearing during the early stages of embryonic development and display a remarkable repertoire of biological functions.6,7 Laminin a4 (LAMA4), a component of Lm-411 and Lm-421, has been found in various tissues of mesenchymal origin, endothelial, and some epithelial basement membranes (BMs).8-10 Numerous analyses indicate that the subunit LAMA4 will dramatically impact the behaviors of endothelial cells via complex interaction with integrin receptors on the surface of endothelial cells.11 However, the role of LAMA4 in the placenta is largely unknown. Therefore, we aimed to explore the effect of LAMA4 during the whole pregnancy and its potential function during development of PE.
1 Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China 2 Laboratory of Lipid & Glucose Research, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
Corresponding Author: Hongbo Qi, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China. Email: [email protected]
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Oxidative stress has been implicated in the pathology of many hypertensive pregnancy-related conditions, including PE and gestational hypertension.12 Reactive oxygen species (ROS) as molecules produced from the reduction in molecular oxygen are the by-products of aerobic respiration and metabolism. These molecules with diverse chemical properties are capable of activating various signaling pathways.13 For instance, the stress-responsive p38 mitogen-activated protein kinase (MAPK) pathway is reported to be robustly activated in PE and is essential to oxidative stress-mediated placental dysfunction.14 It is hypothesized that intermittent placental perfusion, secondary to the deficient trophoblast invasion of endometrial arteries, will induce an ischemia–reperfusion (I/R) insult to the placenta and cause intense oxidative stress in the placental endothelium.15 The disruption of endothelial homeostasis is fundamental to the initiation and progression of PE.16 Mitogen-activated protein kinases cascades transduce a diverse spectrum of extracellular and intracellular stimuli into alterations in gene expression and cellular function. Reports from Reich et al have showed that MAPK are involved in laminin signaling.17 However, few data are available on the potential function of LAMA4 in the process of placental oxidative stress and MAPK signaling pathway. So, we mimicked the pathological condition of PE using a hypoxia–reoxygenation (H/R) circle and utilized RNA interference (RNAi) transfection to further evaluate the effects of LAMA4 on endothelial cell migration and tube formation in vitro. In addition, the p38 MAPK pathway inhibitor SB203580 was applied to study the possible molecular and cellular mechanisms of the LAMA4-p38 MAPK signaling pathway in human umbilical vein endothelial cells (HUVECs). The above-mentioned evidences support the function of LAMA4 in promoting angiogenesis of HUVECs and also suggest that the dysregulation of LAMA4 expression may be associated with the occurrence of PE.
Human placental tissues from 6 to 8 weeks (n ¼ 9) were obtained from healthy women undergoing legal abortion for nonmedical reasons; normal term placentas (n ¼ 15) and PE placentas (n ¼ 17) were collected after cesarean operations. These tissues were collected and washed in 0.9% saline, and then 1 piece of the tissues was fixed in 3% formaldehyde and embedded with paraffin at room temperature for immunohistochemistry (IHC).
Cell Culture and H/R Application The HUVEC cell line was used in our study. The HUVECs were cultured in Dulbecco Modified Eagle Media: Nutrient Mixture F12 (DMEM F12; Gibco, Massachusetts) containing 10% fetal bovine serum (FBS; Gibco). The cells were maintained under standard culture conditions at 37 C under 5%CO2. Then H/R intervention (8 hours under 2% oxygen and 16 hours under standard culture conditions, 2 cycles) was performed in a trigas cell culture incubator at 37 C.
RNA interference and p38 MAPK Inhibition The cells for RNAi were transfected with 100 nmol/L LAMA4 small-interfering RNA (siRNA; 5’-CAGGGAUUUAUGCAGAAAUTT-3’, GenePharma, Shanghai, China; Genbank ID for LAMA4: NM_000006.12) or the control siRNA (a universal negative control, GenePharma) with Lipofectamine 2000 (Invitrogen, Carlsbad, California) as recommended by the manufacturer. Transfection was performed according to the manufacturer’s protocol. The transfection efficiency measured using fluorescence-labeled siRNA was >90%. In order to block the p38 MAPK pathway, SB203580 (Invitrogen, Carlsbad, Calif., USA) was dissolved in dimethyl sulfoxide (DMSO) to 5 mmol/L and then used to preincubate the cells as described previously.18
Immunohistochemistry Method and Material Patients and Tissue Collection The definition and criteria of PE were based on the diagnostic criteria outlined by the American College of Obstetrics and Gynecology. Preeclampsia was characterized by systolic blood pressure 140 mm Hg or diastolic blood pressure 90 mm Hg, together with proteinuria (0.3 g protein in a 24-hour urine collection, usually corresponding to 1 þ on urine dipstick tests) that occurred after 20 weeks of gestation in previously normotensive women. Patients with diabetes mellitus, chronic renal diseases, chronic hypertension, or other metabolic diseases were excluded from this study. This study and the use of samples were conducted under standard experimental protocols approved by the Ethics Committee of Chongqing Medical University. Ethical approval was granted by the Ethics Committee of the First Hospital of Chongqing University. All women were informed and signed consent to donate their placentas for scientific research use. 2
Immunohistochemistry was performed as described previously.19 The sections (each 5 mm thick) were dewaxed, rehydrated, and incubated with 3% H2O2 for 10 minutes to quench the endogenous peroxidase. After blocking with 20% normal goat serum for 30 minutes, the sections were incubated with a primary LAMA4 antibody (1:500) and a platelet endothelial cell adhesion molecule 1 (CD31) antibody (1:500) overnight at 4 C. After washing with phosphate buffer solution (PBS), the sections were incubated with biotinylated secondary antibody and stained using diaminobenzidine solution. Nonimmune rabbit immunoglobulin G was used as a negative control. Each experiment was performed at least three times, and comparable results were obtained each time.
Western Blotting The control and treated cells were homogenized in a lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China), cracked by an ultrasonic probe for 2 minutes and then
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centrifuged at 14 000g at 4 C for 15 minutes to remove the debris. Protein contents were measured using a bicinchoninic acid (BCA) Protein Assay (Beyotime). 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 electro-transferred to polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts). After blockage, the membranes were incubated with appropriate primary antibodies overnight at 4 C, including rabbit antibodies against LAMA4 (1:1000; Sigma-Aldrich Co, St. Louis, Missouri), p38, p-p38, c-Jun NH2-terminal protein kinase (JNK), p-JNK, extracellular signal-regulated kinase (ERK), p-ERK (all 1:1,000; Cell Signaling Technology, Danvers, Massachusetts), and b-actin (1:1000; Santa Cruz Biotechnology, Dallas, Texas). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) at 37 C for 2 hours. Positive bands were detected by enhanced chemiluminescence reagents (Beyotime) and analyzed by Quantity One 4.4.0 (Bio-Rad Laboratories, Hercules, California). Experiments were performed in triplicate.
RNA Extraction and Quantitative Real-Time ReverseTranscriptase Polymerase Chain Reaction Total RNA was extracted from the control and treated cells using a TRIzol regent (Takara Biotechnology, Japan) and purified according to the manufacturer’s instruction. Reverse transcription was performed using a Primescript RT reagent kit (TaKaRa). Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) was performed on a C1000 Thermal Cycler (Bio-Rad). The means of threshold cycles were normalized to atubulin levels, and the relative messenger RNA (mRNA) levels were analyzed using the 2DDCt method. The experiments were performed in triplicate. The primers used in this study include LAMA4 (Forward: 5’-GGAAAATAAGCGAGGCACCG’, Reverse: 5’-AGCCACAGAGGCAGAACCGA-3’) and atubulin (Forward: 5’-CCAA GTTCTGGGAGGTGATCTG 3’, Reverse: 5’- TTGTAGTAGACGTTGATGCGCTC-3’).
Migration Assay The migration assay was performed in 24-well plates (Costar, Cambridge, Massachusetts) and polycarbonate filters (8-mm pores; Transwell Chamber, Millipore) as described previously.20 The HUVECs (1.0 105/well) with or without treatments (including siRNA and SB) were seeded into the upper chamber in a serum-free medium, whereas a medium containing 10% FBS was added to the lower chamber under normoxia at 37 C for 48 hours. The H/R group was differently treated: Normal cells were exposed to H/R under 37 C for 48 hours. Then, the inserts were fixed with methyl alcohol and stained with 3% Crystal violet. The migratory cells were observed and recorded under a microscope (Olympus IX51, Japan) at a magnification of 200 in 10 random fields. The relative migration percentages were calculated with nontreated cells cultured
under normal conditions with an average migration percentage of 100%. All experiments were repeated triple times.
Tube Formation BD Matrigel (cat. no. 356230, BD Biosciences, San Jose, California) was diluted with a serum-free medium at a ratio of 1:2 with cooled pipettes and distributed in a 24-well plate (150 mL/well) on ice. As before, HUVECs (1.0 105/well) with various pretreatments were gently added to each of the triplicate wells after matrigel solidification, followed by H/R intervention or normal culture for 48 hours. Digital images were captured from each well (200). Tube formation was quantified by calculating the total tube length of tube-like structures using a camera (Olympus) interfaced with the software ImageJ (National Institutes of Health, Bethesda, Maryland). The tracks of endothelial cells organizing into cellular cord (tube) networks were counted, and the results from 5 randomly selected fields were averaged. The normalized percentages were calculated using nontreated cells cultured under normal conditions with an average tube formation index [tube length (mm) per mm2] of 100%.
Cell Proliferation Assay After transfection of siRNA, the HUVECs were subjected into 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazo-lium bromide (MTT) assay to test cell proliferation. The HUVECs (5 103/well) were seeded in 96-well plates. After a 20-hour culture, 100 mL of an MTT reagent was added to the medium and gently removed 4 hours later. The DMSO (100 ml) was then added into each well, and the optical density of each well was assessed at wave length 570 nm. Each experiment was performed in triplicate.
Flow Cytometry Analysis of Cell Apoptosis The cell apoptosis rate was measured by an annexin V-fluorescein isothiocyanate (FITC) and PI apoptosis detection kit (Key-GenBiotech, JiangSu, China) as described previously.21 After culture for 48 hours, the cells after various pretreatments were stained with a binding buffer (1 mg/mL propidium iodide and 1 mg/mL FITC-labeled annexin-V) and gently vortexed, then incubated in the dark—at room temperature for 30 minutes. Apoptosis rates were quantified with an FACS Vantage SE Flow Cytometry (FCM) meter (BD Biosciences). The cells that excluded PI and were positive for Annexin V-FITC binding were represented as apoptotic cells.
Immunofluorescence Staining The normal HUVECs and the H/R-intervened cells were permeabilized with 0.2% Triton X-100 and blocked with PBS containing 1% BSA for 1 hour after fixation in ice methanol. An anti-LAMA4 antibody (1:100) was used as the primary antibody. After washing with PBS, the cells were incubated with an FITC secondary antibody (Zhongsan Golden Bridge
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Figure 1. LAMA4 was highly expressed in human endothelial cells during pregnancy. A, Immunostaining of LAMA4 in human villi; its protein was intensely and specifically expressed in syncytotrophoblasts and endothelial cells in the first trimester. B, Expression of CD31 in human villi in the first trimester, it was as a marker of endothelial cells. C, Negative control with nonimmune rabbit IgG in human villi. D, LAMA4 was strongly expressed in human decidua in the first trimester, especially in the endothelial cells and decidual cells. E, Expression of CD31 in human decidua. F, Negative control with nonimmune rabbit IgG in the maternal decidua. G, Expression of LAMA4 was also found in endothelial cells of the normal term placenta. H, LAMA4 was rarely expressed in the placenta of preeclampsia. I, Negative control with nonimmune rabbit IgG in the term placenta. Scale bar ¼ 100 mm. LAMA4 indicates laminin a4; IgG, immunoglobulin.
Crop, Beijing, China) for 1 hour. The nuclei were stained with 3 mg/mL propidium iodide for 8 to 10 minutes. Images were acquired with an Olympus BMX-60 microscope equipped with a cooled charge-coupled device sensi-camera (Cooke, Auburn Hills, Michigan) and the software Slidebook (Intelligent Imaging Innovations, Denver, Colorado). All experiments were repeated in triplicate.
Statistical Analysis Each experiment was performed in triplicate. All the values are expressed as mean + standard error of the mean. The data were analyzed for significance using the software GraphPad Prism (GraphPad Software, San Diego, California). Between the 2 groups, comparisons of continuous variables were conducted using independent t tests. Statistical differences among multiple groups were evaluated by 1-way analysis of 4
variance (ANOVA), followed by the least significant difference multiple comparison as appropriate. P < .05 was considered as significant.
Results 1.
LAMA4 was highly expressed in human endothelial cells during the first trimester.
The LAMA4 protein expressions in human placental villi at different stages of pregnancy were first evaluated using IHC. CD31 was as a marker used to identify endothelial cells (Figures 1B & 1E). During the first trimester, LAMA4 protein was intensely and specifically expressed in syncytiotrophoblasts and endothelial cells (Figures 1A & 1D). Especially, in maternal decidua, LAMA4 was strongly stained in endothelial cells and decidual cells (Figure 1D). In the third trimester,
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Figure 2. Effects of H/R, siRNA, and p38 inhibitor SB203580 on expressions of LAMA4, VEGFR2, and MAPK signaling pathways in HUVECs. A, Protein expressions of LAMA4, VEGFR2, p-p38, and T-p38 (total p38) were examined by Western blotting. B, Statistical analysis of the Western blotting results in (A; n ¼ 3 in triplicate, *P < .05 and **P < .01compare with the control group). C, Representative Western blots of ERK, JNK, p-ERK, and p-JNK proteins in HUVECs with indicated treatments. D, Statistical analysis of the Western blotting results in (C; n ¼ 3 in triplicate, **P < .01compared with the control group). Data were analyzed by ANOVA to assess significant differences. H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; VEGFR2, Vascular endothelial growth factor receptor 2; MAPK, mitogen-activated protein kinase; HUVECs, human umbilical vein endothelial cells; ERK, extracellular signal-regulated kinase; JNK, NH2-terminal protein kinase; p-ERK phophorylated ERK; p-JNK, phophorylated JNK; ANOVA, analysis of variance.
Figure 3. Effects of H/R and siRNA on LAMA4 mRNA expression in HUVECs. A, mRNA expression of LAMA4 was decreased by siRNA transfection examined by RT-qPCR (n ¼ 3 in triplicate, P < .01). B, H/R treatment deduced LAMA4 mRNA expression by almost 60% (n ¼ 3 in triplicate, P < .01). Data were analyzed by t test to assess significant differences. H/R indicates hypoxia–reoxygenation; siRNA, smallinterfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; mRNA, messenger RNA; RT-qPCR, quantitative realtime reverse-transcriptase polymerase chain reaction.
LAMA4 was still expressed in endothelial cells of the normal placentas (Figure 1G) but was even less expressed in PE placentas (Figure 1H). 2.
The LAMA4 siRNA significantly inhibited migration and tube formation of HUVECs.
Given the evident expression of LAMA4 in the firsttrimester placentas, we next investigated whether LAMA4 had
effect on the functions of endothelial cells. We examined the migration or tube formation of HUVECs using siRNA transfection tests. The Western blotting and RT-qPCR showed that siRNA targeting significantly decreased the LAMA4 protein expression in HUVECs (Figures 2A and 3A). Vascular endothelial growth factor receptor 2 (VEGFR2) is expressed most prominently in vascular endothelial cells and their embryonic precursors. This receptor has potent tyrosine kinase activity and can transduce the major signals for
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Figure 4. Migratory ability of HUVECs decreased under the H/R circumstance and LAMA4 downregulation. Representative images showed HUVECs passing through the membrane by migration assay (magnification 200). A-D, Representative images showed HUVECs penetration through the membrane. A, Nontreated cells cultured under normal condition; (B) siRNA group; (C) SB group in which cells were preincubated with SB203580 and cultured under normoxic conditions; (D) H/R group in which cells were maintained under hypoxic conditions for 8 hours followed by reoxygenation for 16 hours, 2 circles. E, Bar graph showed relative numbers of migrating cells (%). Data were analyzed by ANOVA to assess significant differences (n ¼ 3 in triplicate, **P < .01 compared with the control group). H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; ANOVA, analysis of variance; SB, SB203580 4(4– Fluorophenyl)–2–(4–methylsulfinylphenyl)–5–(4–pyridyl) 1 H–imidazole.
angiogenesis.22,23 We tried to investigate the relation between LAMA4 and VEGFR2 expressions and found that the expression levels of VEGFR2 decreased significantly by 1.53-fold after LAMA4 down-regulation (n ¼ 3, P < .001; Figure 2A). Cells after transfection were plated onto a pure filter for determination of migration. Compared with the control group, the LAMA4 siRNA treatment significantly decreased 6
the percentage of cells that migrated to the other side of the filter (n ¼ 3, P < .001, Figures 4A, B, and E). We also examined whether LAMA4 influenced the tube formation in HUVECs. It was noticed that LAMA4 siRNA transfection for 48 hours reduced the tube formation capability of HUVECs (n ¼ 3, p < 0.001, Figures 5A, 5C, & 5E) but not proliferation and/or apoptosis (n ¼ 3, p > 0.05, Figures 6A, B, E, and F).
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Figure 5. Tube formation ability of HUVECs decreased under the H/R circumstance and LAMA4 siRNA transfection. A-D, Representative images revealed tube formation in HUVECs. A, Nontreated group; (B) H/R group; (C) siRNA group; (D) SB group. E, Bar graph showed the normalized tube formation index to the control group in the tube formation assay (%).The tube formation index was expressed as tube length (mm) per area (mm2). Bars ¼ 100 mm. Data were analyzed by ANOVA to assess significant differences (n ¼ 3 in triplicate, **P < .01 compared with the control group). H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; ANOVA, analysis of variance.
3.
The H/R condition suppressed migration and tube formation of HUVECs.
To further detect the potential function of LAMA4 in PE, we used H/R condition to mimic the oxidative stress environment during PE development. Western blotting showed that LAMA4
protein expression was down-regulated by about 92.48% in the HUVECs under H/R treatment compared with the control group (n ¼ 3, P < .001; Figures 2A and 2B). RT-qPCR also consistently showed that LAMA4 mRNA level was reduced after H/R exposure (n ¼ 3, P < .01; Figure 3B). Immunofluorescence also testified that H/R treatment significantly decreased
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Figure 6. The effects of different treatments on apoptosis rates in HUVECs, assessed by flow cytometry. A-D, Representative pictures of flow cytometry for apoptosis rate among normal nontreated HUVECs and different treatments, including H/R, siRNA, and SB. E, MTT assay showed no significant difference in cell proliferation using LAMA4 siRNA treatment in HUVECs. F, LAMA4 siRNA transfection and SB had no significant effects on the apoptosis rate in HUVECs by FCM, but H/R exposure significantly increased the apoptosis rate in HUVECs. Data were analyzed by ANOVA to assess significant differences (n ¼ 3 in triplicate, **P < .01 comparing with the control group). H/R indicates hypoxia–reoxygenation; siRNA, small-interfering RNA; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells; ANOVA, analysis of variance; FCM, flow cytometry; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazo-lium bromide.
the LAMA4 protein expression in HUVECs (Figure 7). Interestingly, consistent with the decline in LAMA4 expression, the VEGFR2 expression level also decreased dramatically after exposure to H/R for 48 hours, tested by Western blotting (n ¼ 3, P < .001, Figure 2A). In addition, our results demonstrated that the migration potential of H/R-treated HUVECs diminished to 50.21% compared with the nontreated cells (n ¼ 3, P < .001, Figures 4A, D, and E). The H/R intervention also reduced the tube formation potential of HUVECs by 43.93% compared to the control group (n ¼ 3, P < .001, Figure 5A, 5B, and 5E). Furthermore, FCM was used to quantify the effect of H/R on apoptosis in HUVECs. A representative FCM image in Figure 6 shows that the H/R environment dramatically promoted the cell apoptosis (n ¼ 3, P < .001; Figures 6A, 6C, and F). 4.
Interactions between MAPK signaling pathway and LAMA4 expression in HUVECs.
The classic oxidative stress-p38 MAPK signaling pathway was selected to explore the mechanism of LAMA4 in 8
HUVECs. Compared with the control group, the p38 inhibitor SB203580 markedly reduced the LAMA4 expression in HUVECs, which was consistent with the reduction in VEGFR2 expression (both n ¼ 3, P < 0.001, Figures 2A & 2B). Moreover, H/R treatment induced significant up-regulation of phosphorylation of p38 (or p-p38) in HUVECs. The bar graphs presenting the quantification of proteins revealed a nearly 2-fold higher level of p-p38 in the H/R group compared with the control group (Figures 2A and B). Since the family of MAPK protein kinases controls many cellular events such as cell proliferation, differentiation, apoptosis, embryogenesis, inflammation, and stress responses,24 we also investigated the expressions of other MAPK proteins after LAMA4 siRNA treatment and H/R exposure, including JNK and ERK. In Figure 2C and D, the results showed that H/R treatment induced higher level of phosphorylation of ERK (p-ERK; n ¼ 3, P < .001) but not phosphorylation of JNK (p-JNK; n ¼ 3, P > .05). However, down-regulation of LAMA4 expression decreased the level of p-JNK (n ¼ 3, P < .01) but not p-ERK (n ¼ 3, P > .05). Our data indicated that although MAPK pathways interacted with LAMAA4, but p38, ERK, and JNK played different roles in regulating
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Figure 7. Immunofluorescent detections of LAMA4 proteins (green) and nuclei (red, propidium iodide, PI) in nontreated and H/R HUVECs. LAMA4 (green) expression decreased in HUVECs after H/R treatment for 48 hours (n ¼ 3 in triplicate). All the pictures were captured 400. /R indicates hypoxia–reoxygenation; LAMA4, laminin a4; HUVECs, human umbilical vein endothelial cells. (The color version of this figure is available in the online version at http://rs.sagepub.com/).
expression of LAMA4 in endothelial cells and responding to oxidative stress.
Discussion The underlying pathophysiology of PE involves the dysregulations of endothelial function in both the maternal and the fetoplacental circulations. Oxidative stress resulting from overproduction of ROS impairs development of the placental vasculature and has been proposed as a key intermediary step during the pathogenesis of PE. The LAMA4 is ubiquitously localized in endothelial BMs throughout various vessels and independent of the developmental stage.25 The LAMA4 is expressed in the mouse developing endothelium from early stages of embryonic development (E8.5).26 In vitro assays suggest several potential roles of LAMA4 in angiogenesis, including cell attachment, cell migration, and tubule formation.27 Our study provided several lines of evidence and demonstrated that LAMA4 promoted the functions of endothelial cells, including migration and angiogenesis. Meanwhile, H/R condition was used to elucidate the underlying oxidative stress-mediated signaling pathways between the effect of LAMA4 and the development of PE. Laminins have been found in the pericellular basement membranes of human decidual cells and placental villi.28 In
this study, LAMA4 was strongly expressed in endothelial cells in placental villi and decidua. The LAMA4 was also highly expressed in the third-trimester placentas compared to the PE placentas. During early pregnancy, decidual angiogenesis forms a new vascular network that serves as the first exchange apparatus between maternal circulation and the developing embryo. Thus, decidual angiogenesis is a fundamental process deciding embryonic survival and successful pregnancy. Our findings suggested that LAMA4 played a role in regulating angiogenesis and placentation in human placentas. Vascular endothelial growth factor plays a key role in decidual angiogenesis and function.29 To further verify the regulatory role of LAMA4 on HUVECs, we investigated the expression of VEGFR2, which was found consistent with the reduction of LAMA4 by siRNA transfection. Downregulation of LAMA4 also decreased cell migration and tube formation potentials in HUVECs. This study proved that LAMA4 may be directly involved in the migration and angiogenesis functions of endothelial cells. To test the effects of long-term H/R on HUVECs and LAMA4, we prolonged the hypoxia to 8 hours and the reoxygenation to 16 hours and extended to 2 circles. The migration and tube formation potentials of HUVECs were dramatically weakened after H/R treatment, accompanied with reduced expressions of LAMA4 and VEGFR2. The above-mentioned evidence strongly suggested that dysregulation of LAMA4 was
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Reproductive Sciences
associated with the impaired angiogenesis function induced by oxidative stress. A growing number of studies show that MAPKs are involved in laminin signaling.17 The MAPK pathways can be activated by a wide variety of different stimuli acting through diverse receptor families and regulated by phosphorylation. The 3 major mammalian MAPK subgroups include p38 MAPK, ERKs, and JNKs/ stress-activated protein kinase (SAPKs).30 The ERK pathway is mainly responsive to mitogens and growth factors and capable of modulating cell survival, migration, and apoptosis.31 The JNK and p38 pathways are activated in response to chemical and environmental stress and to inflammatory cytokines.30 We proved that inhibition of p38 pathway abates the expressions of LAMA4 and VEGFR2 in HUVECs. In addition, p-p38 was expressed at higher level after the H/R treatment, accompanied with the downregulation of LAMA4 expression. Our findings indicated that oxidative stress could lead to p38 activation and abates the impact of LAMA4 in HUVECs. Furthermore, we noticed that p-JNK was reduced by the LAMA4 siRNA transfection, and its activation in HUVECs could be restored by H/R. However, p-ERK was not affected by LAMA4 dysregulation but by H/R exposure. Taken together, our study demonstrated that p38 and JNK pathways directly interacted with LAMA4 expression in endothelial cells, and activation of p38 and ERK pathways induced by oxidative stress were related to down-regulation of LAMA4. These findings indicated that the LAMA4-MAPK pathway is likely to be responsible for the poorly developed fetoplacental vasculature associated with PE. Without doubt, LAMA4 might be regulated by different pathways under the various conditions in endothelial cells, which needs further explorations. The biological effects of laminin are mediated by receptors that are divided into 2 major groups, integrin and nonintegrin receptors. Coregulation and physical association between the 2 receptors are involved in regulating or stabilizing the interaction of laminin.32 For an instance, the interaction of the a6b1 integrin with laminin-8 (LM-411) might promote endothelial cell migration in vivo, since their expression patterns overlap with those of newly formed capillaries, where endothelial cells are actively migrating.33 Hence, further investigations need to carry on exploring the internal interactions between these receptors signaling transductions and LAMA4 function. In summary, our present study supported that LAMA4 is very crucial in promoting the functions of endothelial cells. Oxidative stress plays an important role in controlling the LAMA4 expression. We also demonstrated that MAPK signaling pathways interact with LAMA4 expression in endothelial cells under oxidative stress. These findings provide a new aspect about the cause of PE. Our study represented a good starting point for subsequent studies to further verify the role of LAMA4 in differentiation/ invasion of trophoblasts. Nevertheless, more elaborate models, such as placental explants and PE animal models are needed to more extensively study the role of the LAMA4-MAPK signaling pathways in the development of PE. Further investigations of LAMA4 will provide insight into the establishment of novel diagnostic, therapeutic, and preventative strategies for PE. 10
Acknowledgments We thank Dr Yubin Ding for helping design the article and suggestions. Authors are grateful 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) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article:: This work is supported by National Natural Science Foundation of China grants (nos. 81070502, 81170585, 81100444, 81300509, and 81300508), and the funding of National Key Clinical Department in China.
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