Original Article Gynecol Obstet Invest 2014;78:101–108 DOI: 10.1159/000363294
Received: August 13, 2013 Accepted after revision: April 29, 2014 Published online: July 2, 2014
Tanshinone II-A Is Protective against Human Umbilical Vein Endothelial Cell Injury after Exposure to Serum from Preeclampsia Patients ChunFeng Wu a Jing Yuan a RenFang Sui b ShuYuan Li b JingXia Sun a a b
Department of Obstetrics and Gynecology, First Affiliated Hospital of Harbin Medical University, and Harbin Medical University, Harbin, China
Key Words Preeclampsia · CD40/CD40 ligand · Tanshinone II-A · Human umbilical vein endothelial cells
Abstract Background/Aims: Preeclampsia (PE) is one of the most common and dangerous complications during pregnancy and is characterized by high blood pressure and significant amounts of protein in the urine. Vascular endothelial cell dysfunction is the major pathology in PE. This study was designed to assay the effects of tanshinone II-A (TII-A) on human umbilical vein endothelial cell (HUVEC) injury after incubation with serum from PE patients and to determine the underlying mechanism. Methods: After treating HUVECs with different TII-A concentrations, cell viability, apoptosis and CD40/CD40 ligand (CD40L) mRNA and protein expression levels were measured. Results: Incubation of HUVECs with serum from PE patients induced morphological alterations, caused decreased cell viability and increased the rate of apoptosis. However, TII-A (5–40 μg/ml) significantly reversed these injuries. Importantly, preapplication of TII-A attenuated PE sera-induced expression of CD40 and CD40L mRNA and protein. Conclusion: TII-A has a protective effect against PE sera, likely through regulation of the CD40/CD40L signal transduction pathway. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0378–7346/14/0782–0101$39.50/0 E-Mail [email protected] www.karger.com/goi
Introduction
Preeclampsia (PE), a dangerous pregnancy complication, is very common and characterized by high blood pressure and significant amounts of protein in the urine of a pregnant woman. Substantial correlation of perinatal and neonatal morbidity with PE has been established [1]. However, the exact pathology of this disease is still unknown, and effective therapeutic agents are urgently needed. One factor that has been implicated in endothelial cell dysfunction during PE is the activation of the CD40-CD40 ligand (CD40L) system. CD40 and CD40L, members of the tumor necrosis factor receptor family, are well known for their role in immunity and inflammation [2]. In addition to being expressed by antigen-presenting cells, CD40 is also expressed by endothelial cells, smooth muscle cells, fibroblasts and epithelial cells. Interaction of CD40L with CD40 promotes the generation of several proinflammatory cytokines, including interleukin (IL)-2, interferon-γ, IL-4 and IL-5. Simultaneously, the CD40/ CD40L interaction induces endothelial cells to release IL-6 and IL-8 and to increase active IL-1β expression. Excess active IL-1β may mediate a vascular inflammatory response and increase oxidative stress, subsequently re-
C.W. and J.Y. contributed equally to this work.
JingXia Sun Department of Obstetrics and Gynecology First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street Nangang District, Harbin 150010, Heilongjiang (China) E-Mail sixsw @ 163.com
sulting in vascular endothelial cell injury [3]. For example, CD40/CD40L expression is upregulated in smooth muscle cells and macrophages, which might contribute to the formation and progression of atherosclerosis [4]. Previously, we obtained blood serum directly from PE patients and applied it to human umbilical vein endothelial cells (HUVECs). Importantly, application of PE serum to HUVECs resulted in cell injury and high expression levels of CD40/CD40L [5]. Elevated CD40/CD40L levels may explain, in part, the molecular mechanisms leading to abnormal placentation. Therefore, CD40/ CD40L represents a potential therapeutic target to treat PE. Salvia miltiorrhiza is a Chinese medicine widely used to promote blood flow and treat vascular disease. Tanshinone II-A (TII-A), a fat-soluble compound derived from the root of S. miltiorrhiza Bunge, possesses a range of pharmacological activities, including protection of vascular endothelial cells, expansion of coronary arteries and amelioration of microcirculation [6]. Clinically, TII-A demonstrates cardiovascular and cerebrovascular protection through its antioxidative activity, with few side effects [7–10]. In the present study, we extended our analysis of TII-A to show that TII-A protected against PE through inhibition of apoptosis. Simultaneously, PE sera-induced CD40/CD40L expression was abolished by TII-A preapplication. From our studies, we have demonstrated that TII-A displays anti-PE activity. Materials and Methods Ethics Statement This study was approved by the Institutional Ethics Committee of the First Affiliated Hospital, Harbin Medical University (certification number: 200819; date: September 17, 2008), and patient consent was obtained from each participant. PE Patients and Sera Collection We included 20 patients with PE and 20 randomly selected healthy pregnant (HP) women in this study. Early-onset PE was diagnosed as described by Valensise et al. [11]. The following inclusion criteria for the PE group were used in the present study: gestational age of 20–34 weeks, blood pressure ≥140/90 mm Hg, urine protein ≥300 mg/24 h or immunoreactive urine protein with epigastric pain and headache. The participants were all primiparous with a single fetus, had no history of hypertension or renal disease before pregnancy and were free of placenta previa, early placental abruption or other obstetric complications. Ten milliliters of venous blood was collected from all participants. Serum was isolated by centrifugation at 3,000 rpm for 5 min, after which the supernatant was collected, aliquoted into 1.5-ml Eppendorf tubes and stored at –80 ° C.
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Cell Culture and Drug Treatment HUVECs (ATCC CRL1730 HUV-EC-C, ATCC, Manassas, Va., USA) were cultured in F-12 K medium containing endothelial cell growth supplement (Sciencell, Carlsbad, Calif., USA) and 10% FBS (Gibco, Carlsbad, Calif., USA) in a 5% CO2, 37 ° C incubator. The medium was replaced every second day. After reaching confluency, the cells were subsequently cultured at a 1:2 ratio and passaged every other day. After reaching 80% confluency, the cells were synchronized by starvation in serum-free medium for 24 h. The cells were divided into three groups: (1) a control group with cells cultured in medium containing 10% maternal serum obtained from HP women; (2) a PE model group with cells cultured in medium containing 10% maternal serum obtained from PE patients, and (3) a TII-A group where cells were pretreated with 5.0, 10.0, 20.0 or 40.0 μg/ml TII-A for 24 h, then cultured in medium containing 10% maternal serum obtained from PE patients for an additional 24 h. TII-A, purchased from National Institutes for Food and Drug Control (China, Lot No. 110766-200619), was diluted in DMSO and stored at 4 ° C in a 40 μg/μl stock solution. The final concentration of DMSO in the treatment group was less than 0.1%.
MTT Assay HUVECs were seeded onto 96-well plates at a density of 3 × 103 cells/ml. After 24 h of incubation, the cells were cultured in serumfree medium for 24 h. After the indicated treatment, 20 μl of MTT (5 mg/ml) was added into each well and incubated with the cells for 4 h before the supernatant was discarded. DMSO (150 μl) was added before shaking for 10 min until crystals were fully dissolved. Absorbance was measured at 490 nm using a TECAN microplate reader (Männedorf, Switzerland). Apoptosis Detection After the cells were treated with normal or PE sera in the presence or absence of TII-A (methods described above), HUVECs were digested with trypsin. The cells were centrifuged at 1,000 rpm for 5 min and washed with PBS. Apoptosis was assayed using the Annexin V-FITC/PI apoptosis assay kit (Neobioscience, Shenzhen, China). Before measurement, 5 μl of annexin V and 10 μl of propidium iodide (PI) were added to the cells for 10 min. Flow cytometry (BD Biosciences) was used to measure apoptosis in the different treatment conditions. Three negative control groups were included, as follows: (1) no dye, (2) annexin V alone and (3) PI alone. CD40 and CD40L Expression After reaching 80% confluency in 6-well plates, HUVECs were digested with trypsin and collected by centrifugation at 1,000 rpm for 5 min before washing twice with PBS to remove cell debris. The cells were resuspended in F-12 K culture medium containing 10% FBS, and the cell concentration was adjusted to 5 × 105 cells/ml. Direct fluorescence detection was performed by incubating 100 μl of staining buffer (PBS with 2% fetal calf serum, 0.1% NaN3 and 5 μl of FITC-conjugated antiCD40 or CD40L monoclonal antibody; BD Biosciences, USA) for 30 min at 4 ° C before centrifugation at 1,000 rpm for 5 min. The supernatant was discarded, and the cells were washed twice with cold PBS prior to centrifugation at 1,000 rpm for 5 min. After fixation with 500 μl of 1% paraformaldehyde, CD40/CD40L
Wu/Yuan/Sui/Li/Sun
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Fig. 1. Effects of TII-A on PE sera-induced morphological alterations of HUVECs. a Representative image of HUVECs after incubation with HP serum for 24 h. b Representative image of HUVECs after treatment with PE sera. c Representative image of HUVECs pretreated with TII-A before PE sera exposure. ×200.
expression was determined using flow cytometry (BD Biosciences) by counting 10,000 cells. Mean fluorescence intensity was used to measure the level of CD40 and CD40L protein expression in HUVECs relative to the standard normal reference fluorescence intensity (in candelas, the international unit of light intensity). RT-PCR Analysis mRNA from each group was isolated using a TRIzol kit (Molecular Research Center Inc., Cincinnati, Ohio, USA). cDNA was obtained by reverse transcription using the Roche First-Strand cDNA Synthesis Kit (San Francisco, Calif., USA). In addition to a dNTP mix (TaKaRa Biotechnology, Dalian, China) and Taq enzyme (TaKaRa), the following PCR primers were designed by Primer 5 software and synthesized by Invitrogen (Shanghai, China): CD40 (124 bp), forward 5′-TTGGTGGTGGTGGTG TTG-3′, and reverse 5′-GCATCTGTGTATATGGCTTCC-3′; CD40L (213 bp), forward 5′-CCTCTGCCACCTTCTCTG-3′, and reverse 5′-TCTTCTATCTTGTCCAACCTTC-3′, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 202 bp), forward 5′-ACGGATTTGGTCGTATTGGG-3′, and reverse 5′-TCCTGG AAGATGGTGATGGG-3′. GAPDH mRNA expression was used as an internal reference. The PCR amplification reaction conditions were as follows: predenaturation at 94 ° C for 3 min, 30 amplification cycles at 94 ° C for 30 s, 63 ° C for 30 s (for CD40) or 60 ° C for 30 s (for CD40L), and 72 ° C for 30 s, with a final extension at 72 ° C for 7 min. Agarose gel electrophoresis (2%) was imaged using an Alpha Innotech imaging system (Johannesburg, South Africa). Density scanning of the electrophoretic bands was performed, and relative target mRNA expression was determined using the gray value of CD40 or CD40L divided by that of GAPDH.
Statistical Methods Statistical analyses were performed using SPSS 13 statistical software (IBM, Chicago, Ill., USA). All data are expressed as means and standard deviation from 6 repeated experiments. Differences among means were considered significant at a value of p < 0.05. Student’s t test was used to analyze the difference between two groups. One-way analysis of variance was used to analyze the differences between multiple groups.
Effect of Tanshinone II-A on a HUVEC Model of Preeclampsia
Table 1. Clinical information on the study participants
Age, years Body weight at sampling, kg Height, m Body mass index, kg/m2
HP group (n = 20)
PE group (n = 20)
p value
27.50±2.82 69.25±7.20 1.61±0.04 26.69±2.90
28.65±3.20 70.10±6.80 1.60±0.03 27.36±2.96
0.235 0.703 0.383 0.477
Results
Clinical Information on the Study Participants Forty pregnant women, including 20 patients with PE (PE group) and 20 HP women (HP group), were included in this study. As shown in table 1, no significant differences in age, body weight, height or body mass index were found between the two groups. Effects of TII-A on PE Sera-Induced Morphological Alterations of HUVECs Initially, we determined the influence of patient sera on HUVEC morphology using inverted microscopy. HUVECs cultured in the presence of HP maternal sera were arranged in a mosaic-like monolayer with cells appearing round and flat or polygonal with a cobblestone appearance (fig. 1a). Culturing HUVECs in PE maternal sera significantly perturbed cell morphology. Specifically, the cells were sparsely distributed with blurred boundaries between the nucleus and the cytoplasm, and dark cytoplasmic granules were also observed (fig. 1b). Strikingly, pretreatment with TII-A (40 mg/ml) could rescue the cells from PE sera-induced morphological alterations. Similar to the HP group, cells in the TII-A group were polygonal or oval shaped and arranged tightly with clear cell boundaries (fig. 1c). Gynecol Obstet Invest 2014;78:101–108 DOI: 10.1159/000363294
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apoptosis induced by PE sera treatment (5 μg/ml TII-A + PE: 9.74 ± 0.70%; 10 μg/ml TII-A + PE: 9.40 ± 0.70%; 20 μg/ml TII-A + PE: 7.47 ± 0.86%; 40 μg/ml TII-A + PE: 5.24 ± 0.98%). Effects of TII-A on the PE Sera-Induced Increase in CD40 and CD40L Expression We next examined CD40 and CD40L expression at the protein and mRNA levels in the different treatment conditions using flow cytometry and RT-PCR analyses, respectively. As shown in figure 4, PE sera treatment significantly upregulated CD40 and CD40L protein expression in HUVECs when compared with the HP control group (p < 0.05). Pretreatment with TII-A significantly reversed the observed increase in CD40 and CD40L expression in comparison with the PE sera-treated group. Importantly, the effect of TII-A was concentration dependent. Analysis of transcript levels by RT-PCR showed a similar increase in CD40 and CD40L mRNA expression levels after treatment with PE sera when compared with the HP control group (p < 0.05). Preapplication of different concentrations of TII-A prevented an increase in CD40 and CD40L levels (fig. 5).
Discussion
Effects of TII-A on the PE Sera-Induced Decrease in HUVEC Viability Given that pretreatment of cells with TII-A prevented PE sera-induced morphological changes, we next sought to determine whether cell viability was also affected. Compared with maternal sera from the HP control group, the sera from the PE group significantly decreased HUVEC viability (p < 0.05; fig. 2). However, pretreatment with TII-A significantly prevented the PE sera-induced decrease in cell viability in a concentration-dependent manner. Thus, pretreatment with TII-A had a protective effect in our HUVEC PE model. Effects of TII-A on PE Sera-Induced HUVEC Apoptosis Because decreased HUVEC viability caused by PE sera could be prevented with TII-A pretreatment, we next investigated whether apoptosis was similarly affected. Compared with the HP control group (1.41 ± 0.09%), incubation with PE sera significantly increased the HUVEC apoptosis rate to 12.09 ± 0.91%, as measured by flow cytometry (fig. 3). Preapplication of different concentrations of TII-A significantly reduced the rate of 104
Gynecol Obstet Invest 2014;78:101–108 DOI: 10.1159/000363294
Without treatment, PE can develop into eclampsia, a life-threatening condition that causes seizures during pregnancy. Currently, the only effective method to relieve PE is pregnancy termination. However, early pregnancy termination to alleviate PE increases both neonatal mortality and morbidity. Magnesium sulfate, which causes prostaglandin release and subsequent dilation of blood vessels, has been occasionally used as a therapeutic drug for PE [12–14]. Despite extending gestational age to some extent, magnesium sulfate does not reverse the development of PE but rather alleviates symptoms temporarily. In endothelial cells, the oxidation reaction is known to regulate normal physiological activity, such as vascular inflammation, low-density lipoprotein oxidation and decreased nitric oxide bioactivity [15]. However, overactivation of the oxidation reaction leads to oxidative stress. Experimental evidence implicates oxidative stress-induced endothelial dysfunction as the major pathology in PE [16–18]. For example, activation of the Gadd45/P38 MAPK pathway can cause an increase in oxidative stress, which might lead to PE [19]. Therefore, agents with the potential to abrogate oxidative stress may prevent or improve PE. Wu/Yuan/Sui/Li/Sun
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Fig. 3. a–f Effect of TII-A on PE sera-induced apoptosis of HUVECs detected by flow cytometry with annexin V-FITC/PI staining. a HP sera. b PE sera. c 5 μg/ml TII-A + PE. d 10 μg/ml TII-A + PE.
e 20 μg/ml TII-A + PE. f 40 μg/ml TII-A + PE. g Pooled data of the apoptosis rate from the different groups. a p < 0.05 compared with HP sera; b p < 0.05 compared with PE serum.
Effect of Tanshinone II-A on a HUVEC Model of Preeclampsia
Gynecol Obstet Invest 2014;78:101–108 DOI: 10.1159/000363294
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CD40L expression. a p < 0.05 compared with HP sera; b p < 0.05 compared with PE sera.
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Wu/Yuan/Sui/Li/Sun
Finding effective Chinese medicines to treat PE is a new idea that has garnered extensive attention in China. Among the putative medicines, S. miltiorrhiza is a strong candidate. It has been shown that a combination of Astragalus membranaceus and S. miltiorrhiza could treat early serious eclampsia by decreasing arterial pressure, reducing 24-hour urinary protein excretion and blood viscosity, ameliorating microcirculation and extending gestational age [20]. Additionally, a single application of S. miltiorrhiza or application of S. miltiorrhiza in combination with low-molecular-weight heparin could significantly extend gestational age and reduce urinary protein excretion [21]. Despite these initial studies on the effect of S. miltiorrhiza on PE symptom alleviation, the molecular mechanisms underlying the therapeutic effects of Salvia are still poorly understood. TII-A is one of the most abundant constituents of the root of S. miltiorrhiza and exerts antioxidant and anti-inflammatory actions in many experimental disease models, especially models of endothelial injury [9, 22]. For example, TII-A administration repaired vascular endothelial injury through decreased lipid peroxidation and regulation of endothelial dilatation [7, 23]. Using a HUVEC peroxide injury model, Lin et al. [10] demonstrated that TII-A exerted its protection through maintenance of cellular superoxide dismutase activity, which antagonized oxidative stress. Experiments have shown that TII-A can prevent peroxide-induced macrophage damage by increasing glutathione peroxidase levels [22]. Consistent with these reports, our data clearly demonstrated that TII-A rescued cell viability and attenuated apoptosis in the presence of PE sera. Importantly, we determined that TII-A exerted its vascular endothelial protection through inhibition of CD40 and CD40L expression. Clinical research demonstrated that elevated CD40/CD40L expression is tightly correlated
with the development of PE; increased CD40/CD40L expression has been observed in platelets and mononuclear cells of PE patients [24]. Lievens et al. [25] found that platelet CD40L strengthened the interaction between platelets and white blood cells and platelets and endothelial cells and mediated leukocyte recruitment through CC chemokine ligand 2. These reactions ultimately caused thrombus and inflammation in atherosclerosis patients. Similarly, we previously verified that CD40/CD40L expression in peripheral blood and umbilical cord blood collected from PE patients was higher than that in blood from HP women [26]. By modeling HUVEC injury via PE sera application, we consistently saw that PE sera increased CD40 and CD40L expression, which led to HUVEC apoptosis [5]. The data presented here further emphasize the critical roles of CD40/CD40L expression levels during PE development. Using our previously established HUVEC injury model, we applied different concentrations of TII-A. The PE sera-induced increase in CD40/CD40L was abolished by TII-A treatment at the mRNA and protein levels.
Conclusion
In conclusion, this study demonstrates that TII-A protection against PE sera-induced HUVEC injury occurs through attenuation of CD40/CD40L expression. Though we provide clues for the mechanism underlying the protective effect of TII-A against PE, uncovering the exact molecular pathway warrants further investigation. Disclosure Statement The authors declare that they have no conflicts of interest to disclose.
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Wu/Yuan/Sui/Li/Sun
Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Received: August 13, 2013 Accepted after revision: April 29, 2014 Published online: July 2, 2014
Tanshinone II-A Is Protective against Human Umbilical Vein Endothelial Cell Injury after Exposure to Serum from Preeclampsia Patients ChunFeng Wu a Jing Yuan a RenFang Sui b ShuYuan Li b JingXia Sun a a b
Department of Obstetrics and Gynecology, First Affiliated Hospital of Harbin Medical University, and Harbin Medical University, Harbin, China
Key Words Preeclampsia · CD40/CD40 ligand · Tanshinone II-A · Human umbilical vein endothelial cells
Abstract Background/Aims: Preeclampsia (PE) is one of the most common and dangerous complications during pregnancy and is characterized by high blood pressure and significant amounts of protein in the urine. Vascular endothelial cell dysfunction is the major pathology in PE. This study was designed to assay the effects of tanshinone II-A (TII-A) on human umbilical vein endothelial cell (HUVEC) injury after incubation with serum from PE patients and to determine the underlying mechanism. Methods: After treating HUVECs with different TII-A concentrations, cell viability, apoptosis and CD40/CD40 ligand (CD40L) mRNA and protein expression levels were measured. Results: Incubation of HUVECs with serum from PE patients induced morphological alterations, caused decreased cell viability and increased the rate of apoptosis. However, TII-A (5–40 μg/ml) significantly reversed these injuries. Importantly, preapplication of TII-A attenuated PE sera-induced expression of CD40 and CD40L mRNA and protein. Conclusion: TII-A has a protective effect against PE sera, likely through regulation of the CD40/CD40L signal transduction pathway. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0378–7346/14/0782–0101$39.50/0 E-Mail [email protected] www.karger.com/goi
Introduction
Preeclampsia (PE), a dangerous pregnancy complication, is very common and characterized by high blood pressure and significant amounts of protein in the urine of a pregnant woman. Substantial correlation of perinatal and neonatal morbidity with PE has been established [1]. However, the exact pathology of this disease is still unknown, and effective therapeutic agents are urgently needed. One factor that has been implicated in endothelial cell dysfunction during PE is the activation of the CD40-CD40 ligand (CD40L) system. CD40 and CD40L, members of the tumor necrosis factor receptor family, are well known for their role in immunity and inflammation [2]. In addition to being expressed by antigen-presenting cells, CD40 is also expressed by endothelial cells, smooth muscle cells, fibroblasts and epithelial cells. Interaction of CD40L with CD40 promotes the generation of several proinflammatory cytokines, including interleukin (IL)-2, interferon-γ, IL-4 and IL-5. Simultaneously, the CD40/ CD40L interaction induces endothelial cells to release IL-6 and IL-8 and to increase active IL-1β expression. Excess active IL-1β may mediate a vascular inflammatory response and increase oxidative stress, subsequently re-
C.W. and J.Y. contributed equally to this work.
JingXia Sun Department of Obstetrics and Gynecology First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street Nangang District, Harbin 150010, Heilongjiang (China) E-Mail sixsw @ 163.com
sulting in vascular endothelial cell injury [3]. For example, CD40/CD40L expression is upregulated in smooth muscle cells and macrophages, which might contribute to the formation and progression of atherosclerosis [4]. Previously, we obtained blood serum directly from PE patients and applied it to human umbilical vein endothelial cells (HUVECs). Importantly, application of PE serum to HUVECs resulted in cell injury and high expression levels of CD40/CD40L [5]. Elevated CD40/CD40L levels may explain, in part, the molecular mechanisms leading to abnormal placentation. Therefore, CD40/ CD40L represents a potential therapeutic target to treat PE. Salvia miltiorrhiza is a Chinese medicine widely used to promote blood flow and treat vascular disease. Tanshinone II-A (TII-A), a fat-soluble compound derived from the root of S. miltiorrhiza Bunge, possesses a range of pharmacological activities, including protection of vascular endothelial cells, expansion of coronary arteries and amelioration of microcirculation [6]. Clinically, TII-A demonstrates cardiovascular and cerebrovascular protection through its antioxidative activity, with few side effects [7–10]. In the present study, we extended our analysis of TII-A to show that TII-A protected against PE through inhibition of apoptosis. Simultaneously, PE sera-induced CD40/CD40L expression was abolished by TII-A preapplication. From our studies, we have demonstrated that TII-A displays anti-PE activity. Materials and Methods Ethics Statement This study was approved by the Institutional Ethics Committee of the First Affiliated Hospital, Harbin Medical University (certification number: 200819; date: September 17, 2008), and patient consent was obtained from each participant. PE Patients and Sera Collection We included 20 patients with PE and 20 randomly selected healthy pregnant (HP) women in this study. Early-onset PE was diagnosed as described by Valensise et al. [11]. The following inclusion criteria for the PE group were used in the present study: gestational age of 20–34 weeks, blood pressure ≥140/90 mm Hg, urine protein ≥300 mg/24 h or immunoreactive urine protein with epigastric pain and headache. The participants were all primiparous with a single fetus, had no history of hypertension or renal disease before pregnancy and were free of placenta previa, early placental abruption or other obstetric complications. Ten milliliters of venous blood was collected from all participants. Serum was isolated by centrifugation at 3,000 rpm for 5 min, after which the supernatant was collected, aliquoted into 1.5-ml Eppendorf tubes and stored at –80 ° C.
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Cell Culture and Drug Treatment HUVECs (ATCC CRL1730 HUV-EC-C, ATCC, Manassas, Va., USA) were cultured in F-12 K medium containing endothelial cell growth supplement (Sciencell, Carlsbad, Calif., USA) and 10% FBS (Gibco, Carlsbad, Calif., USA) in a 5% CO2, 37 ° C incubator. The medium was replaced every second day. After reaching confluency, the cells were subsequently cultured at a 1:2 ratio and passaged every other day. After reaching 80% confluency, the cells were synchronized by starvation in serum-free medium for 24 h. The cells were divided into three groups: (1) a control group with cells cultured in medium containing 10% maternal serum obtained from HP women; (2) a PE model group with cells cultured in medium containing 10% maternal serum obtained from PE patients, and (3) a TII-A group where cells were pretreated with 5.0, 10.0, 20.0 or 40.0 μg/ml TII-A for 24 h, then cultured in medium containing 10% maternal serum obtained from PE patients for an additional 24 h. TII-A, purchased from National Institutes for Food and Drug Control (China, Lot No. 110766-200619), was diluted in DMSO and stored at 4 ° C in a 40 μg/μl stock solution. The final concentration of DMSO in the treatment group was less than 0.1%.
MTT Assay HUVECs were seeded onto 96-well plates at a density of 3 × 103 cells/ml. After 24 h of incubation, the cells were cultured in serumfree medium for 24 h. After the indicated treatment, 20 μl of MTT (5 mg/ml) was added into each well and incubated with the cells for 4 h before the supernatant was discarded. DMSO (150 μl) was added before shaking for 10 min until crystals were fully dissolved. Absorbance was measured at 490 nm using a TECAN microplate reader (Männedorf, Switzerland). Apoptosis Detection After the cells were treated with normal or PE sera in the presence or absence of TII-A (methods described above), HUVECs were digested with trypsin. The cells were centrifuged at 1,000 rpm for 5 min and washed with PBS. Apoptosis was assayed using the Annexin V-FITC/PI apoptosis assay kit (Neobioscience, Shenzhen, China). Before measurement, 5 μl of annexin V and 10 μl of propidium iodide (PI) were added to the cells for 10 min. Flow cytometry (BD Biosciences) was used to measure apoptosis in the different treatment conditions. Three negative control groups were included, as follows: (1) no dye, (2) annexin V alone and (3) PI alone. CD40 and CD40L Expression After reaching 80% confluency in 6-well plates, HUVECs were digested with trypsin and collected by centrifugation at 1,000 rpm for 5 min before washing twice with PBS to remove cell debris. The cells were resuspended in F-12 K culture medium containing 10% FBS, and the cell concentration was adjusted to 5 × 105 cells/ml. Direct fluorescence detection was performed by incubating 100 μl of staining buffer (PBS with 2% fetal calf serum, 0.1% NaN3 and 5 μl of FITC-conjugated antiCD40 or CD40L monoclonal antibody; BD Biosciences, USA) for 30 min at 4 ° C before centrifugation at 1,000 rpm for 5 min. The supernatant was discarded, and the cells were washed twice with cold PBS prior to centrifugation at 1,000 rpm for 5 min. After fixation with 500 μl of 1% paraformaldehyde, CD40/CD40L
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Fig. 1. Effects of TII-A on PE sera-induced morphological alterations of HUVECs. a Representative image of HUVECs after incubation with HP serum for 24 h. b Representative image of HUVECs after treatment with PE sera. c Representative image of HUVECs pretreated with TII-A before PE sera exposure. ×200.
expression was determined using flow cytometry (BD Biosciences) by counting 10,000 cells. Mean fluorescence intensity was used to measure the level of CD40 and CD40L protein expression in HUVECs relative to the standard normal reference fluorescence intensity (in candelas, the international unit of light intensity). RT-PCR Analysis mRNA from each group was isolated using a TRIzol kit (Molecular Research Center Inc., Cincinnati, Ohio, USA). cDNA was obtained by reverse transcription using the Roche First-Strand cDNA Synthesis Kit (San Francisco, Calif., USA). In addition to a dNTP mix (TaKaRa Biotechnology, Dalian, China) and Taq enzyme (TaKaRa), the following PCR primers were designed by Primer 5 software and synthesized by Invitrogen (Shanghai, China): CD40 (124 bp), forward 5′-TTGGTGGTGGTGGTG TTG-3′, and reverse 5′-GCATCTGTGTATATGGCTTCC-3′; CD40L (213 bp), forward 5′-CCTCTGCCACCTTCTCTG-3′, and reverse 5′-TCTTCTATCTTGTCCAACCTTC-3′, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 202 bp), forward 5′-ACGGATTTGGTCGTATTGGG-3′, and reverse 5′-TCCTGG AAGATGGTGATGGG-3′. GAPDH mRNA expression was used as an internal reference. The PCR amplification reaction conditions were as follows: predenaturation at 94 ° C for 3 min, 30 amplification cycles at 94 ° C for 30 s, 63 ° C for 30 s (for CD40) or 60 ° C for 30 s (for CD40L), and 72 ° C for 30 s, with a final extension at 72 ° C for 7 min. Agarose gel electrophoresis (2%) was imaged using an Alpha Innotech imaging system (Johannesburg, South Africa). Density scanning of the electrophoretic bands was performed, and relative target mRNA expression was determined using the gray value of CD40 or CD40L divided by that of GAPDH.
Statistical Methods Statistical analyses were performed using SPSS 13 statistical software (IBM, Chicago, Ill., USA). All data are expressed as means and standard deviation from 6 repeated experiments. Differences among means were considered significant at a value of p < 0.05. Student’s t test was used to analyze the difference between two groups. One-way analysis of variance was used to analyze the differences between multiple groups.
Effect of Tanshinone II-A on a HUVEC Model of Preeclampsia
Table 1. Clinical information on the study participants
Age, years Body weight at sampling, kg Height, m Body mass index, kg/m2
HP group (n = 20)
PE group (n = 20)
p value
27.50±2.82 69.25±7.20 1.61±0.04 26.69±2.90
28.65±3.20 70.10±6.80 1.60±0.03 27.36±2.96
0.235 0.703 0.383 0.477
Results
Clinical Information on the Study Participants Forty pregnant women, including 20 patients with PE (PE group) and 20 HP women (HP group), were included in this study. As shown in table 1, no significant differences in age, body weight, height or body mass index were found between the two groups. Effects of TII-A on PE Sera-Induced Morphological Alterations of HUVECs Initially, we determined the influence of patient sera on HUVEC morphology using inverted microscopy. HUVECs cultured in the presence of HP maternal sera were arranged in a mosaic-like monolayer with cells appearing round and flat or polygonal with a cobblestone appearance (fig. 1a). Culturing HUVECs in PE maternal sera significantly perturbed cell morphology. Specifically, the cells were sparsely distributed with blurred boundaries between the nucleus and the cytoplasm, and dark cytoplasmic granules were also observed (fig. 1b). Strikingly, pretreatment with TII-A (40 mg/ml) could rescue the cells from PE sera-induced morphological alterations. Similar to the HP group, cells in the TII-A group were polygonal or oval shaped and arranged tightly with clear cell boundaries (fig. 1c). Gynecol Obstet Invest 2014;78:101–108 DOI: 10.1159/000363294
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apoptosis induced by PE sera treatment (5 μg/ml TII-A + PE: 9.74 ± 0.70%; 10 μg/ml TII-A + PE: 9.40 ± 0.70%; 20 μg/ml TII-A + PE: 7.47 ± 0.86%; 40 μg/ml TII-A + PE: 5.24 ± 0.98%). Effects of TII-A on the PE Sera-Induced Increase in CD40 and CD40L Expression We next examined CD40 and CD40L expression at the protein and mRNA levels in the different treatment conditions using flow cytometry and RT-PCR analyses, respectively. As shown in figure 4, PE sera treatment significantly upregulated CD40 and CD40L protein expression in HUVECs when compared with the HP control group (p < 0.05). Pretreatment with TII-A significantly reversed the observed increase in CD40 and CD40L expression in comparison with the PE sera-treated group. Importantly, the effect of TII-A was concentration dependent. Analysis of transcript levels by RT-PCR showed a similar increase in CD40 and CD40L mRNA expression levels after treatment with PE sera when compared with the HP control group (p < 0.05). Preapplication of different concentrations of TII-A prevented an increase in CD40 and CD40L levels (fig. 5).
Discussion
Effects of TII-A on the PE Sera-Induced Decrease in HUVEC Viability Given that pretreatment of cells with TII-A prevented PE sera-induced morphological changes, we next sought to determine whether cell viability was also affected. Compared with maternal sera from the HP control group, the sera from the PE group significantly decreased HUVEC viability (p < 0.05; fig. 2). However, pretreatment with TII-A significantly prevented the PE sera-induced decrease in cell viability in a concentration-dependent manner. Thus, pretreatment with TII-A had a protective effect in our HUVEC PE model. Effects of TII-A on PE Sera-Induced HUVEC Apoptosis Because decreased HUVEC viability caused by PE sera could be prevented with TII-A pretreatment, we next investigated whether apoptosis was similarly affected. Compared with the HP control group (1.41 ± 0.09%), incubation with PE sera significantly increased the HUVEC apoptosis rate to 12.09 ± 0.91%, as measured by flow cytometry (fig. 3). Preapplication of different concentrations of TII-A significantly reduced the rate of 104
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Without treatment, PE can develop into eclampsia, a life-threatening condition that causes seizures during pregnancy. Currently, the only effective method to relieve PE is pregnancy termination. However, early pregnancy termination to alleviate PE increases both neonatal mortality and morbidity. Magnesium sulfate, which causes prostaglandin release and subsequent dilation of blood vessels, has been occasionally used as a therapeutic drug for PE [12–14]. Despite extending gestational age to some extent, magnesium sulfate does not reverse the development of PE but rather alleviates symptoms temporarily. In endothelial cells, the oxidation reaction is known to regulate normal physiological activity, such as vascular inflammation, low-density lipoprotein oxidation and decreased nitric oxide bioactivity [15]. However, overactivation of the oxidation reaction leads to oxidative stress. Experimental evidence implicates oxidative stress-induced endothelial dysfunction as the major pathology in PE [16–18]. For example, activation of the Gadd45/P38 MAPK pathway can cause an increase in oxidative stress, which might lead to PE [19]. Therefore, agents with the potential to abrogate oxidative stress may prevent or improve PE. Wu/Yuan/Sui/Li/Sun
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Fig. 3. a–f Effect of TII-A on PE sera-induced apoptosis of HUVECs detected by flow cytometry with annexin V-FITC/PI staining. a HP sera. b PE sera. c 5 μg/ml TII-A + PE. d 10 μg/ml TII-A + PE.
e 20 μg/ml TII-A + PE. f 40 μg/ml TII-A + PE. g Pooled data of the apoptosis rate from the different groups. a p < 0.05 compared with HP sera; b p < 0.05 compared with PE serum.
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Gynecol Obstet Invest 2014;78:101–108 DOI: 10.1159/000363294
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Fig. 4. Effects of TII-A on the PE sera-induced increase in CD40 and CD40L expression detected by flow cytometry. The mean fluorescence intensity of each group was analyzed for CD40 (a) and CD40L (b). a p < 0.05 compared with HP sera; b p < 0.05 compared with PE sera; c p < 0.05 compared with 5.0 μg/ml TII-A + PE.
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CD40L expression. a p < 0.05 compared with HP sera; b p < 0.05 compared with PE sera.
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Wu/Yuan/Sui/Li/Sun
Finding effective Chinese medicines to treat PE is a new idea that has garnered extensive attention in China. Among the putative medicines, S. miltiorrhiza is a strong candidate. It has been shown that a combination of Astragalus membranaceus and S. miltiorrhiza could treat early serious eclampsia by decreasing arterial pressure, reducing 24-hour urinary protein excretion and blood viscosity, ameliorating microcirculation and extending gestational age [20]. Additionally, a single application of S. miltiorrhiza or application of S. miltiorrhiza in combination with low-molecular-weight heparin could significantly extend gestational age and reduce urinary protein excretion [21]. Despite these initial studies on the effect of S. miltiorrhiza on PE symptom alleviation, the molecular mechanisms underlying the therapeutic effects of Salvia are still poorly understood. TII-A is one of the most abundant constituents of the root of S. miltiorrhiza and exerts antioxidant and anti-inflammatory actions in many experimental disease models, especially models of endothelial injury [9, 22]. For example, TII-A administration repaired vascular endothelial injury through decreased lipid peroxidation and regulation of endothelial dilatation [7, 23]. Using a HUVEC peroxide injury model, Lin et al. [10] demonstrated that TII-A exerted its protection through maintenance of cellular superoxide dismutase activity, which antagonized oxidative stress. Experiments have shown that TII-A can prevent peroxide-induced macrophage damage by increasing glutathione peroxidase levels [22]. Consistent with these reports, our data clearly demonstrated that TII-A rescued cell viability and attenuated apoptosis in the presence of PE sera. Importantly, we determined that TII-A exerted its vascular endothelial protection through inhibition of CD40 and CD40L expression. Clinical research demonstrated that elevated CD40/CD40L expression is tightly correlated
with the development of PE; increased CD40/CD40L expression has been observed in platelets and mononuclear cells of PE patients [24]. Lievens et al. [25] found that platelet CD40L strengthened the interaction between platelets and white blood cells and platelets and endothelial cells and mediated leukocyte recruitment through CC chemokine ligand 2. These reactions ultimately caused thrombus and inflammation in atherosclerosis patients. Similarly, we previously verified that CD40/CD40L expression in peripheral blood and umbilical cord blood collected from PE patients was higher than that in blood from HP women [26]. By modeling HUVEC injury via PE sera application, we consistently saw that PE sera increased CD40 and CD40L expression, which led to HUVEC apoptosis [5]. The data presented here further emphasize the critical roles of CD40/CD40L expression levels during PE development. Using our previously established HUVEC injury model, we applied different concentrations of TII-A. The PE sera-induced increase in CD40/CD40L was abolished by TII-A treatment at the mRNA and protein levels.
Conclusion
In conclusion, this study demonstrates that TII-A protection against PE sera-induced HUVEC injury occurs through attenuation of CD40/CD40L expression. Though we provide clues for the mechanism underlying the protective effect of TII-A against PE, uncovering the exact molecular pathway warrants further investigation. Disclosure Statement The authors declare that they have no conflicts of interest to disclose.
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