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Inhibition of endocan attenuates monocrotaline-induced connective tissue disease related pulmonary arterial hypertension
International Immunopharmacology 42 (2017) 115–121
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Inhibition of endocan attenuates monocrotaline-induced connective tissue disease related pulmonary arterial hypertension Haiyan Zhao a, Yunxin Xue b, Yun Guo a, Yue Sun a, Dongmei Liu a, Xiaofei Wang a,⁎ a b
Department of Immunology and Rheumatology, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China Department of Respiration, Liaoning Jinqiu Hospital, Shenyang 110016, People's Republic of China
a r t i c l e
i n f o
Article history: Received 11 July 2016 Received in revised form 31 October 2016 Accepted 18 November 2016 Available online xxxx Keywords: Endocan Pulmonary arterial hypertension Endothelial cells TNF-α MAPK
a b s t r a c t Connective tissue disease related pulmonary arterial hypertension (CTD-PAH) is characterized by vascular remodeling, endothelial dysfunction and inflammation. Endocan is a novel endothelial dysfunction marker. The aim of the present study was to investigate the role of endocan in CTD-PAH. Monocrotaline (MCT)-induced PAH rats were used as the CTD-PAH model. Short hairpin RNA packed in a lentiviral vector used to inhibit endocan expression was intratracheally instilled in rats prior to the MCT injection. Endocan was found to be increased in the serum and lung of MCT-induced PAH rats. Short hairpin RNA mediated knockdown of endocan significantly decreased right ventricular systolic pressure, attenuated pulmonary remodeling and inflammatory responses in the lung. In the in vitro study, tumor necrosis factor-α (TNF-α) exposure caused increased endocan expression in the primary cultured rat pulmonary microvascular endothelial cells (RPMECs). Endocan knockdown inhibited the permeability increase and adhesion molecules secretion in RPMECs induced by TNF-α. In addition, TNF-α induced MAPK activation was blocked when endocan gene was knocked down. These data demonstrate that endocan may play an important role in the development of CTD-PAH. This study provides novel evidence to better understand the pathogenesis of CTD-PAH, which may be beneficial for the treatment of this disease. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Pulmonary arterial hypertension (PAH) is a syndrome characterized by increased mean pulmonary arterial pressure (mPAP). PAH develops pulmonary vessel remodeling and changes the features of hemodynamic, and finally leads to right heart failure. Connective tissue disease (CTD) is closely associated with PAH. Patients with CTD are at increased risk for developing PAH [1]. The prevalence of PAH in systemic sclerosis, a CTD that is most commonly associated with PAH, is 7.85–13% of patients [2–4]. Moreover, CTD-related PAH (CTD-PAH) has a poor prognosis. In China, the survival rates of patients with CTD-PAH are inferior to those of patients with idiopathic PAH [5]. However, the pathogenesis of CTD-PAH has not been fully revealed yet. To investigate the possible molecular mechanisms of CTD-PAH would be beneficial for the treatment of this disease. Endothelial cell (EC) dysfunction is one of the pathological processes that contribute to the development of PAH. ECs are essential for vascular structure and functions. For example, ECs produce a number of cytokines that regulate the physical functions of pulmonary vessels, such ⁎ Corresponding author at: Department of Immunology and Rheumatology, Shengjing Hospital of China Medical University, No. 36 Sanhao Street, Heping District, Shenyang, Liaoning 110004, People's Republic of China. E-mail address: [email protected] (X. Wang).
http://dx.doi.org/10.1016/j.intimp.2016.11.016 1567-5769/© 2016 Elsevier B.V. All rights reserved.
as angiogenesis, vasoconstriction and vasodilatation. EC dysfunction results in imbalance of these cytokines, induces vasoconstriction and smooth muscle cells hypertrophy, and finally leads to vascular remodeling [6]. ECs also protect the smooth muscle cells and fibroblasts of the vessel from exposing to injurious factors. Damage of EC barrier integrity leads to vascular tissue cells expose to the excessive cytokines induced by stimulus and facilitates the injury. Increasing evidence has accumulated indicating that inflammation plays an integral role in the pathogenesis of PAH [7]. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine secreted by macrophages in response to inflammatory stimuli. Mice with overexpression of TNFα showed right ventricle hypertrophy and pulmonary hypertension [8], and TNF-α antagonist etanercept attenuate monocrotaline (MCT)-induced PAH [9,10]. In addition, TNF-α also plays a key role in the progress of CTD. TNF-α antagonists are widely used in patients with various CTD such as arthritis, systemic sclerosis, and ankylosing spondylitis [11–14]. Endocan, previously known as endothelial cell specific molecule-1 (ESM-1), is a soluble dermatan sulfate proteoglycan which was first cloned from human umbilical vein endothelial cell (HUVEC) cDNA library in 1996 [15]. Endocan serves as a regulator of vascular cell processes such as proliferation, adhesion and migration [15,16]. Endocan is considered as a novel EC dysfunction marker because it is mainly secreted by EC and its alteration often occurs in conditions characterized
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by endothelial dysfunction. Till now, dysregulation of endocan has been found in cancers [17,18], sepsis [19], cardiovascular diseases [20,21] and diabetic retinopathy [22]. However, at best of our knowledge, no study has reported the role of endocan in CTD-PAH. In the present study, virus packed endocan- short hairpin RNA (shRNA) was injected into MCT-induced rats, a CTD-PAH model, to investigate the role of endocan in this disease. In addition, TNF-α-induced primary pulmonary microvascular endothelial cells were also used to investigate possible underlying mechanisms in vitro. 2. Materials and methods
2.4. ELISA Lung tissue and primary endothelial cells were homogenized in cool PBS and repeated freezing in liquid nitrogen and thawing. The homogenate was then centrifuged at 12,000 g for 10 min at 4 °C. Protein concentrations in the supernatants were determined using a BCA protein assay kit (Beyotime). The levels of endocan and TNF-α in the serum, lung tissue or cell culture medium were analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Boster, Wuhan, China) following the manufactures' instructions.
2.1. Animals 2.5. Isolation and culture of RPMECs All animal studies were approved by the ethics committee of the China Medical University. Male Sprague-Dawley (SD) rats weighing 180–220 g were obtained from Laboratory Animal Center of China Medical University, (Shenyang, China). The rats were maintained in a temperature and humidity-controlled room (21– 22 °C, 75– 80%) with a 12/12-h light/dark cycle and had free access to standard rat chow and water ad libitum. The animals were randomly divided into: 1) control group (Con, n = 10); 2) MCT-induced pulmonary arterial hypertension group (PAH, n = 10); 3) PAH and scramble shRNA group (PAH + scr shRNA, n = 10); and 4) PAH and endocan shRNA group (PAH + endo shRNA, n = 10). The EGFP-encoding lentiviral strain carrying the shRNA oligonucleotides that target 5′-GGTGACGAGTTTGGTGTC-3′ on endocan mRNA or a scramble shRNA with the sequence of 5′TTCTCCGAACGTGTCACGT-3′ were obtained from Hanbio Co., Ltd. (Shanghai, China). Endocan shRNA or scramble shRNA was were administrated to the rats in the shRNA groups by intratracheal instillation through the mouth daily for 6 days (in total 1.5 × 108 transducing units in 300 μl PBS, 50 μl per day). Rats in the PAH groups received a single subcutaneous injection of 60 mg/kg MCT (Meilun, Dalian, China) which was dissolved in a mixed solution of ethanol and saline with the volume ratio of 2:8, at day 7. Rats in the control group received the same volume of vehicle. The rats were maintained for another 21 days. The blood was collected at day 1, 7, 14 and 21 after MCT injection.
Rat pulmonary microvascular endothelial cells (RPMECs) were isolated from pulmonary arteries as previously described [23]. Briefly, male SD rats (300–350 g) were anesthetized by an intraperitoneal injection of pentobarbital sodium. After thoracotomy, the pulmonary vasculature was perfused by injection of ice-cold PBS into the right ventricle. Lungs were then removed and placed in ice-cold serum-free Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA, USA). Thin strips were removed from the outermost surface of the lung periphery, minced and digested using 3% type II collagenase (Sigma-Aldrich, St Louis, MO, USA). The mixture was filtered through a 100 μm mesh, centrifuged at 300 ×g, and washed twice with cold PBS. The cells were resuspended in RPMI 1640 supplemented with 10% FBS (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin.
2.6. Inhibiting endocan expression in vitro Primary cultured RPMECs were infected with either Lenti-EGFPendocan-shRNA or Lenti-EGFP-scramble shRNA for 12, 24 and 48 h. The infection efficiency was examined by real time PCR and Western blotting.
2.7. Permeability assay in vitro 2.2. Right ventricular systolic pressure (RVSP) measurement At day 22, the rats were anesthetized intraperitoneally with 50 mg/kg of pentobarbital (i.p.). A heparin (0.3%) filled polyethylene catheter was introduced into the right ventricle through the right jugular vein. The catheter was connected to a BL-420F biological data acquisition and analysis system (Chengdu Techman Software Co., Ltd., Chengdu, China) using a pressure transducer. The digitalized RVSP was recorded. After the hemodynamic measurement, the rats were euthanized and the blood and lung tissue were collected for further analysis.
Permeability of RPMECs was quantitated spectrophotometrically by measuring the flux of Evans blue-bound albumin across RPMEC monolayers. RPMEC layer were incubated with indicated concentration of endocan in transwell chamber for 6 h. The cells were then washed with PBS for 3 times. Subsequently, fresh culture medium was added to the lower chamber, and the medium contain 0.67 mg/ml Evens blue and 4% BSA was added in the upper chamber. The optical density at 630 nm was measured in the lower chamber after 10 min incubation.
2.8. RNA isolation and quantitative real-time PCR 2.3. Histological analysis For lentiviral transduction efficiency determination in the lung, the lungs were perfused with PBS and fixed in 4% paraformaldehyde for 2 h. Next, the fixed lung tissues were frozen and cut into 10-μm-thick sections. The nucleoli were stained using 4′, 6-diamidino-2phenylindole (DAPI) and the intensity of EGFP was observed under a fluorescence microscope (BX53, Olympus, Tokyo, Japan). For H&E staining, the lung tissues were fixed in 4% paraformaldehyde overnight and embedded in paraffin. The paraffin blocks were cut into 5-μm-thick sections and stained with hematoxylin and eosin (H&E, Solarbio Science & Technology, Co., Ltd., Beijing, China). The sections were examined under a light microscopy. The index of pulmonary arterial wall thickness was calculated as the following formula: (external diameter-internal diameter) / external diameter × 100%.
Total RNA was isolated from lung tissue or RPMECs using a RNAsimple Total RNA Kit (TIANGEN Biotech, Beijing, China) following the manufacture's protocol. Complementary DNA (cDNA) was generated from total RNA using oligo-dT and Super Moloney Murine Leukemia Virus Reverse Transcriptase (BioTeke, Beijing, China). Real-time PCR reactions were each performed in a total volume of 20 μl reaction mixture, containing 1 μl cDNA, 10 μl 2 × SYBR Green Master Mix (BioTeke, Beijing, China), and 0.5 μl of each primer on an Exicycler 96 fluorescence quantitative detector (Bioneer, Daejeon, Korea). The transcript number was calculated using a 2−ΔΔCt method with β-actin as an internal reference. The primer sequences were listed as following: endocan: forward: 3′-TTCGGTGACGAGTTTGGTG-5′, reverse: 3′-TGTTTGGGAGGCAGAGGT5′; β-actin: forward: 3′-GGAGATTACTGCCCTGGCTCCTAGC-5′, reverse: 3′-GGCCGGACTCATCGTACTCCTGCTT-5′.
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2.9. Western blotting analysis Lung tissue or RPMECs were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). Total protein was extracted and concentration was determined using a Bicinchoninic Acid Protein Assay Kit (Beyotime). Total protein was separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis and the target proteins were transfer to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C and incubated with goat anti-rabbit IgG-HRP (1:5000; Beyotime) at room temperature for 45 min. Immunoreactive bands were visualized using enhanced chemiluminecent (7 Sea Pharmtech, Shanghai, China) and exposed on films (Fuji Photo Film, Tokyo, Japan). The gray values of blots were analyzed using a Gel-Pro-Analyzer software (Media Cybernetics, Rockville, MD, USA). The primary antibodies used in this study were as following: endocan antibody (1:500, PAC463Ra01, USCN Life Science, Wuhan, China), VCAM1 antibody (1:400, BA3840, Boster), ICAM1 antibody (1:400, PB0054, Boster) , E-selecin antibody (1:400, BA0615, Boster), ERK antibody (1:500, bs-2637R, Bioss, Beijing, China), pERK antibody (1:500, bs-1522R, Bioss), JNK antibody (1:500, bs-10562R, Bioss), pJNK antibody (1:500, bs-1640R, Bioss), p38 antibody (1:500, bs0637R, Bioss), and p-p38 antibody (1:500, bs-5477R, Bioss). 2.10. Statistical analysis Data were presented as the mean ± standard deviation (SD). Statistical tests were performed using SPSS software (19.0, IBM, New York, NY, USA). Comparisons between mean values of multiple groups were performed using one-way analysis of variance (ANOVA), followed by Fisher's least significant difference (LSD) post hoc test for multiple comparisons. P b 0.05 indicates statistically significant difference. 3. Results 3.1. shRNA mediated endocan inhibition in vivo Rat pulmonary tissues were observed under a fluorescence microscope 21 days after intratracheal gene transfer with Lenti-EGFP. Strong green fluorescence was observed in the pulmonary arterioles of rats from Lenti-EGFP treated groups, while no fluorescence was found in
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those of rats treated with PBS (Fig. 1A). In addition, endocan mRNA and protein levels were dramatically decreased in the lung of rats in the endocan-shRNA treated group (Fig. 1B and C). The scrambleshRNA has no effect on endocan expression. 3.1. Endocan inhibition attenuated right ventricular dysfunction and pulmonary inflammation Endocan expression level was measured in the lungs of MCT-exposed rats. As shown in Fig. 2A, the level of endocan in the lung was increased with time after treatment of MCT. The mRNA expression level of endocan was also significantly increased in the lungs of MCT-exposted rats (Fig. 1B). MCT induced an obvious increase in RVSP (36.03 ± 7.54 vs. 18.91 ± 3.17 mmHg, PAH vs. normal, P b 0.01, Fig. 2B). Endocan-shRNA administration in PAH rats resulted in significant attenuation of RVSP (27.3 ± 7.29 mm Hg, P b 0.01 vs. PAH). In addition, MCT exposure resulted in inflammatory response in the lungs as evidenced by significantly increased level of TNF-α (Fig. 2C), and this was suppressed by the inhibition of endocan. 3.2. Endocan inhibition attenuated pulmonary vessel remodeling The remodeling of the pulmonary arterial wall was determined. The lung tissue sections were stained using H&E and the pulmonary arterial wall thickness was calculated. In agreement with a previous study [24], MCT resulted in a significant increase in the index of pulmonary arterial wall thickness (38.1 ± 8.8% vs. 16.1 ± 4.2% PAH vs. normal, P b 0.01), which indicated a severe pulmonary arterial wall remodeling (Fig. 3). After inhibition of endocan expression by RNA interference, the thickening of the arterial wall induced by MCT was attenuated (21.5 ± 5.6%, P b 0.01 vs. PAH). 3.3. Effects of endocan inhibition on TNF-α-exposed RPMECs Endocan-shRNA-mediated RNAi were tested in RPMECs in vitro. Endocan knockdown induced a time-dependent decrease in mRNA and protein expression of endocan (Fig. 4A and B). The asynchronous expression of mRNA and protein may because the protein expression was later. Endocan-shRNA treated for 48 h was used in the following studies. TNF-α plays a key role in the progression of CTDs, including CTD-associated PAH [9]. Therefore, TNF-α-exposed RPMECs were used as an in
Fig. 1. Inhibition of endocan expression in vivo. Rat pulmonary tissues were observed under a fluorescence microscope after intratracheal gene transfer with Lenti-EGFP or PBS. Green fluorescence was found in the lung tissues of rats received Lenti6.3-EGFP-endocan-shRNA or Lenti-EGFP intratracheally but not in the rats received PBS (A). Results of quantitative real-time PCR (B) and western blot analysis (C) showed the decreased expression of endocan in the pulmonary tissues of endocan-shRNA treated rats. Values were represented as means ± SD (n = 5 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the PAH group. 1: Normal; 2: PAH; 3: PAH + endocan-shRNA; 4: PAH + scr-shRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Effects of endocan inhibition on PAH. Serum endocan was increased in MCT-induced PAH rats (A). Values were represented as means ± SD (n = 10 in each group). **P b 0.01 compared with the normal group at the same day; #P b 0.05 compared with the PAH group at day 1; ##P b 0.01 compared with the PAH group at day 1. Endocan knockdown decreased right ventricular systolic pressure (B) and reduced tumor necrosis factor-α level in the lung (C). Values were represented as means ± SD (n = 10 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the PAH group.
vitro model of CTD-associated PAH. As shown in Fig. 4C and D, TNF-α 20 ng/ml induced a markedly increase of endocan mRNA and protein expression in cultured RPMECs, which was consistent with the previous study [25]. To investigate the effect of endocan in TNF-α-exposed RPMECs, endocan expression was inhibited using endocan-shRNA. The decreased mRNA and protein expression of endocan indicated the success of the RNA interference. As illustrated in Fig. 4E and F, endocan administration induced a dose-dependent increase in permeability of RPMECs. In addition, endocan knockdown significantly inhibited TNF-α-induced permeability increase in RPMECs. 3.4. Effects of endocan on adhesion molecules expression in RPMECs The expression of the adhesion molecules on ECs is an important event in vascular inflammatory responses. In the present study, the expression levels of VCAM-1, ICAM-1 and E-selectin were detected in endocan-exposed RPMECs. As presented in Fig. 5A–D, endocan resulted in dose-dependently increases in the protein expression levels of VCAM-1, ICAM-1 and E-selectin. In addition, TNF-α also induced marked increases of the protein expression levels of VCAM-1, ICAM-1 and Eselectin in endothelial cell (Fig. 5E–H). However, shRNA-mediated endocan inhibition completely blocked the effects of TNF-α on the expression of these adhesion molecules, which indicated TNF-α may be involved in endocan-induced adhesion molecules expression in RPMECs. 3.5. Effects of endocan on the MAPK signaling pathway in RPMECs The MAPK signaling pathway is essential in TNF-α-induced ECs [26, 27], and also plays important roles in the pulmonary arterial ECs of MCT-induced PAH rats [28]. In this study, increased phosphorylation
levels of ERK, JNK and p38 was found in endocan-exposed RPMECs (Fig. 6A–C). TNF-α also caused significant increases in the phosphorylation levels of the MAPK proteins (Fig. 6D–F). Inhibiting endocan by shRNA completely prevented TNF-α-induced MAPK phosphorylation. 4. Discussion Endocan has been demonstrated to be abnormally expressed in various diseases, including cancer, cardiovascular diseases, and diabetes [17–22]. As an endothelial cell marker, endocan was reported to be involved in endothelial dysfunction and angiogenesis in these diseases. CTD-PAH is characterized by endothelial dysfunction and pulmonary arterial remodeling. Thus, the present study investigated the role of endocan in a rat model of CTD-PAH. The results demonstrated that endocan expression and serum endocan levels were increased in MCT-induced CTD-PAH rats. Short hairpin RNA-mediated endocan inhibition attenuated syndrome of CTD-PAH in the rats. In addition, TNF-α also resulted in an increase of endocan expression in the cultured RPMECs in vitro, and endocan knockdown inhibited the increase of endothelial permeability and adhesion molecules expression induced by TNF-α. Moreover, the MAPK signaling pathway may be involved in the pathological mechanisms of endocan. Among the animal models that have been developed for the study of PAH pathogenesis, MCT-induced PAH rat model was believed to be the model that could better mimic the progression and the inflammatory environment of CTD-PAH [9,29]. In the present study, the rats exposure to MCT showed significantly increased RVSP and TNF-α level in the lung, and obvious pulmonary arterial remodeling, which was consistent with the previous studies [30,31]. In addition, we found, for the first time, the serum level of endocan in MCT-induced PAH rats were increased. Considering the important role of endocan in endothelial functions, it is possibly involved in the endothelial dysfunction of CTD-PAH.
Fig. 3. Effects of endocan inhibition on pulmonary arterial remodeling in PAH rats. Lung tissue sections were stained using hematoxylin and eosin. Compared to tissue from normal rats (A), PAH rats showed thickened arterial wall (B), and shRNA-mediated endocan knockdown reversed this change (C). Scramble shRNA showed no effect on pulmonary arterial wall of PAH rats. The index of wall thickness was calculated and showed in (E). Scale bar: 100 μM. Values were represented as means ± SD (n = 10 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the PAH group.
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Fig. 4. Effects of endocan inhibition on TNF-α-exposed RPMECs. shRNA-meidated endocan RNAi time-dependently diminished endocan mRNA (A) and protein (B) expression in PRMECs. **P b 0.01 compared with the scr-shRNA group in the same time. TNF-α upregulated the mRNA (C) and protein (D) expression of endocan in RPMECs, and this was inhibited by endocan knockdown. Endocan induced a dose-dependent increase of permeability in RPMECs (E). Endocan knockdown inhibited TNF-α-induced increase of permeability in RPMECs (F). Values were represented as means ± SD (n = 5 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the TNF-α group. Numbers in panel B: 1, normal cells; 2, 4, 6, endo-shRNA 12, 24 and 48 h; 3, 5, 7, scr-shRNA 12, 24, and 48 h. Numbers in panel D: 1, normal cells; 2, TNF-α; 3, TNF-α + endo-shRNA; 4, TNF-α + scr-shRNA.
Moreover, shRNA mediated endocan gene knockdown decreased RVSP and pulmonary arterial remodeling, which confirmed the roles of endocan played in CTD-PAH.
In MCT-induced PAH model, inflammatory cells infiltrate into the lung tissues and secrete various inflammatory cytokines including TNF-α [32]. TNF-α also plays an important role in CTD. In the present
Fig. 5. Effects of endocan on the secretion of adhesion molecules in RPMECs. Endocan dose-dependently increased the protein expression of VCAM-1 (B), ICAM-1 (C) and E-selectin (D). Endocan knockdown inhibited the increased VCAM-1 (F), ICAM-1 (G) and E-selectin (H) induced by TNF-α. The reprehensive protein blots were shown in A and E. Values were represented as means ± SD (n = 3). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the TNF-α group.
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Fig. 6. Effects of endocan on the MAPK signaling pathway in RPMECs. Endocan induced phosphorylation of ERK (A), JNK (B) and p38 (C) in PRMECs. Endocan knockdown inhibited TNF-αinduced activation of MAPK proteins (D–F). Values were represented as means ± SD (n = 3). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the TNF-α group. 1: Normal; 2: TNF-α; 3: TNF-α + endocan-shRNA; 4: TNF-α + scr-shRNA.
study, MCT-induced CTD-PAH rats showed increased TNF-α expression in the lung, and endocan knockdown reduced this increase. In the in vitro study, TNF-α induced upregulation of endocan expression in primary RPMECs, and endocan knockdown attenuated TNF-α-induced injury in the RPMECs. These results indicate that in the CTD-PAH there may be a reciprocal mechanism between endocan and TNF-α. These two cytokines can stimulate the expression of each other, and both of them contribute to the endothelial dysfunction. Therefore, endocan knockdown decreased the expression endocan itself and reduced the increased TNF-α induced by endocan, which attenuated endothelial dysfunction, and finally improved symptoms of PAH. As discussed above, inflammatory processes is the common characteristic of PAH and CTD. In addition to inflammatory cytokines like TNFα, vascular cell adhesion molecules also play important roles in inflammatory processes. Endothelial injury induces secretion of adhesion molecules and the excessive adhesion molecules cause further endothelial damage by facilitating leukocyte adhesion and migration to endothelium [33,34]. In the present study, the expression of VCAM-1, ICAM-1, and Eselectin was investigated. These adhesion molecules have been found to
be increased in CTD-PAH patients [35] and MCT-induced PAH models [36,37]. In addition, endocan upregulates the expression of these three cell adhesion molecules in ECs [38]. Our results demonstrated that endocan dose-dependently increased the expression of VCAM-1, ICAM1, and E-selectin in primary RPMECs, and endocan knockdown inhibited the expression of the three adhesion molecules induced by TNF-α in vitro. These results suggest that the expression of cell adhesion molecules regulated by endocan may be involved in the pathological mechanisms of CTD-PAH. The MAPK family includes signaling molecules that regulate various physiological and pathological processes, including cellular proliferation, differentiation, inflammatory responses and apoptosis. In response to stimuli, these signaling molecules are phosphorylated and further activate target genes or proteins. MAPK signaling has been found to be related with pathological processes in PAH such as pulmonary vascular remodeling and inflammatory responses [39,40]. Treatment with p38 MAPK inhibitor lowered the mean pulmonary artery pressure and reduced right ventricular hypertrophy in MCT-induced PAH rats [39]. Considering TNF-α exposure can induce MAPK phosphorylation, excessive
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TNF-α may contribute to the activation of MAPK in CTD-PAH. In the present study, endocan resulted in phosphorylation of ERK, JNK and p38, the three primary proteins in the MAPK family, and endocan knockdown completely blocked the effects of TNF-α on MAPKs. This result indicate that TNF-α activates MAPK through regulating endocan expression. In a previous study on RKO colon cancer cells [41], endocan expression was significantly increased in ERK2 knockdown cells. In our study, the reverse effect of MAPK on endocan has not been investigated. Whether there is feedback mechanism between MAPK and endocan in CTD-PAH need to be further studied. In summary, our study demonstrated that levels of endocan in the serum and lung were increased in MCT-induced CTD-PAH rats, and endocan knockdown by shRNA effectively attenuated PAH symptoms and reduced inflammatory responses in the lung. These results suggest that endocan may play an important role in the development of CTDPAH. This study provides novel evidence to better understand the pathogenesis of CTD-PAH, which may be beneficial for the treatment of this disease. Acknowledgements This study was supported by a grant from the Diagnostic and Treatment Capability Construction Project of Key Clinical Specialty for the Reform Programme of Liaoning Provincial Hospital (No. LNCCC-D 37-2015).
[17]
[18]
[19] [20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
References [1] Y.K. Sung, L. Chung, Connective tissue disease-associated pulmonary arterial hypertension, Rheum. Dis. Clin. N. Am. 41 (2015) 295–313. [2] D. Mukerjee, D. St George, B. Coleiro, C. Knight, C.P. Denton, J. Davar, et al., Prevalence and outcome in systemic sclerosis associated pulmonary arterial hypertension: application of a registry approach, Ann. Rheum. Dis. 62 (2003) 1088–1093. [3] E. Hachulla, V. Gressin, L. Guillevin, P. Carpentier, E. Diot, J. Sibilia, et al., Early detection of pulmonary arterial hypertension in systemic sclerosis: a French nationwide prospective multicenter study, Arthritis Rheum. 52 (2005) 3792–3800. [4] S. Phung, G. Strange, L.P. Chung, J. Leong, B. Dalton, J. Roddy, et al., Prevalence of pulmonary arterial hypertension in an Australian scleroderma population: screening allows for earlier diagnosis, Intern. Med. J. 39 (2009) 682–691. [5] R. Zhang, L.Z. Dai, W.P. Xie, Z.X. Yu, B.X. Wu, L. Pan, et al., Survival of Chinese patients with pulmonary arterial hypertension in the modern treatment era, Chest 140 (2011) 301–309. [6] M. Mandegar, Y.C. Fung, W. Huang, C.V. Remillard, L.J. Rubin, J.X. Yuan, Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension, Microvasc. Res. 68 (2004) 75–103. [7] M. Humbert, N.W. Morrell, S.L. Archer, K.R. Stenmark, M.R. MacLean, I.M. Lang, et al., Cellular and molecular pathobiology of pulmonary arterial hypertension, J. Am. Coll. Cardiol. 43 (2004) 13S–24S. [8] M. Fujita, J.M. Shannon, C.G. Irvin, K.A. Fagan, C. Cool, A. Augustin, et al., Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension, Am. J. Physiol. Lung Cell. Mol. Physiol. 280 (2001) L39–L49. [9] L.L. Zhang, J. Lu, M.T. Li, Q. Wang, X.F. Zeng, Preventive and remedial application of etanercept attenuate monocrotaline-induced pulmonary arterial hypertension, Int. J. Rheum. Dis. 19 (2016) 192–198. [10] Q. Wang, X.R. Zuo, Y.Y. Wang, W.P. Xie, H. Wang, M. Zhang, Monocrotaline-induced pulmonary arterial hypertension is attenuated by TNF-alpha antagonists via the suppression of TNF-alpha expression and NF-kappaB pathway in rats, Vasc. Pharmacol. 58 (2013) 71–77. [11] T. Cobo-Ibanez, M.A. Descalzo, E. Loza-Santamaria, L. Carmona, S. Munoz-Fernandez, Serious infections in patients with rheumatoid arthritis and other immune-mediated connective tissue diseases exposed to anti-TNF or rituximab: data from the Spanish registry BIOBADASER 2.0, Rheumatol. Int. 34 (2014) 953–961. [12] A. Saraux, J. Benichou, L. Guillevin, L. Idbrik, C. Job-Deslandre, J. Sibilia, et al., Which patients with rheumatoid arthritis, spondyloarthritis, or juvenile idiopathic arthritis receive TNF-alpha antagonists in France? The CORPUS cohort study, Clin. Exp. Rheumatol. 33 (2015) 602–610. [13] G. Murdaca, F. Spano, M. Contatore, A. Guastalla, F. Puppo, Potential use of TNF-alpha inhibitors in systemic sclerosis, Immunotherapy 6 (2014) 283–289. [14] L.J. Maxwell, J. Zochling, A. Boonen, J.A. Singh, M.M. Veras, E. Tanjong Ghogomu, et al., TNF-alpha inhibitors for ankylosing spondylitis, Cochrane Database of Syst. Rev. 4 (2015), CD005468. [15] P. Lassalle, S. Molet, A. Janin, J.V. Heyden, J. Tavernier, W. Fiers, et al., ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines, J. Biol. Chem. 271 (1996) 20458–20464. [16] D. Bechard, A. Scherpereel, H. Hammad, T. Gentina, A. Tsicopoulos, M. Aumercier, et al., Human endothelial-cell specific molecule-1 binds directly to the integrin
[28]
[29]
[30]
[31]
[32]
[33]
[34] [35]
[36]
[37]
[38] [39]
[40]
[41]
121
CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1, J. Immunol. 167 (2001) 3099–3106. N. Liu, L.H. Zhang, H. Du, Y. Hu, G.G. Zhang, X.H. Wang, et al., Overexpression of endothelial cell specific molecule-1 (ESM-1) in gastric cancer, Ann. Surg. Oncol. 17 (2010) 2628–2639. L.Y. Chen, X. Liu, S.L. Wang, C.Y. Qin, Over-expression of the endocan gene in endothelial cells from hepatocellular carcinoma is associated with angiogenesis and tumour invasion, J. Int. Med. Res. 38 (2010) 498–510. C. Palmiere, M. Augsburger, Endocan measurement for the postmortem diagnosis of sepsis, Legal Med. 16 (2014) 1–7. S. Balta, D.P. Mikhailidis, S. Demirkol, C. Ozturk, E. Kurtoglu, M. Demir, et al., Endocan–a novel inflammatory indicator in newly diagnosed patients with hypertension: a pilot study, Angiology 65 (2014) 773–777. X.S. Wang, W. Yang, T. Luo, J.M. Wang, Y.Y. Jing, Serum endocan levels are correlated with the presence and severity of coronary artery disease in patients with hypertension, Genet. Test. Mol. Biomarkers 19 (2015) 124–127. A.M. Abu El-Asrar, M.I. Nawaz, G. De Hertogh, A.S. Al-Kharashi, K. Van den Eynde, G. Mohammad, et al., The angiogenic biomarker endocan is upregulated in proliferative diabetic retinopathy and correlates with vascular endothelial growth factor, Curr. Eye Res. 40 (2015) 321–331. J. King, T. Hamil, J. Creighton, S. Wu, P. Bhat, F. McDonald, et al., Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes, Microvasc. Res. 67 (2004) 139–151. L.Q. Bi, R. Zhu, H. Kong, S.L. Wu, N. Li, X.R. Zuo, et al., Ruscogenin attenuates monocrotaline-induced pulmonary hypertension in rats, Int. Immunopharmacol. 16 (2013) 7–16. D. Bechard, V. Meignin, A. Scherpereel, S. Oudin, G. Kervoaze, P. Bertheau, et al., Characterization of the secreted form of endothelial-cell-specific molecule 1 by specific monoclonal antibodies, J. Vasc. Res. 37 (2000) 417–425. H. Choi, H.N. Nguyen, F.S. Lamb, Inhibition of endocytosis exacerbates TNF-alpha-induced endothelial dysfunction via enhanced JNK and p38 activation, Am. J. Physiol. Heart Circ. Physiol. 306 (2014) H1154–H1163. W. Olejarz, D. Bryk, D. Zapolska-Downar, M. Malecki, A. Stachurska, D. Sitkiewicz, Mycophenolic acid attenuates the tumour necrosis factor-alpha-mediated proinflammatory response in endothelial cells by blocking the MAPK/NF-kappaB and ROS pathways, Eur. J. Clin. Investig. 44 (2014) 54–64. A.C. Church, D.H. Martin, R. Wadsworth, G. Bryson, A.J. Fisher, D.J. Welsh, et al., The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension, Am. J. Physiol. Lung Cell. Mol. Physiol. 309 (2015) L333–L347. D.W. Wilson, H.J. Segall, L.C. Pan, S.K. Dunston, Progressive inflammatory and structural changes in the pulmonary vasculature of monocrotaline-treated rats, Microvasc. Res. 38 (1989) 57–80. A. Csiszar, N. Labinskyy, S. Olson, J.T. Pinto, S. Gupte, J.M. Wu, et al., Resveratrol prevents monocrotaline-induced pulmonary hypertension in rats, Hypertension 54 (2009) 668–675. Y. Sadamura-Takenaka, T. Ito, S. Noma, Y. Oyama, S. Yamada, K. Kawahara, et al., HMGB1 promotes the development of pulmonary arterial hypertension in rats, PLoS One 9 (2014), e102482. T.M. Kolb, R.L. Damico, P.M. Hassoun, Linking new and old concepts: inflammation meets the Warburg phenomenon in pulmonary arterial hypertension, J. Mol. Med. 89 (2011) 729–732. C.J. Kirkpatrick, M. Wagner, I. Hermanns, C.L. Klein, H. Kohler, M. Otto, et al., Physiology and cell biology of the endothelium: a dynamic interface for cell communication, Int. J. Microcirc. Clin. Exp. 17 (1997) 231–240. J. Panes, M. Perry, D.N. Granger, Leukocyte-endothelial cell adhesion: avenues for therapeutic intervention, Br. J. Pharmacol. 126 (1999) 537–550. F. Iannone, M.T. Riccardi, S. Guiducci, R. Bizzoca, M. Cinelli, M. Matucci-Cerinic, et al., Bosentan regulates the expression of adhesion molecules on circulating T cells and serum soluble adhesion molecules in systemic sclerosis-associated pulmonary arterial hypertension, Ann. Rheum. Dis. 67 (2008) 1121–1126. J.D. Roberts Jr., J.D. Chiche, J. Weimann, W. Steudel, W.M. Zapol, K.D. Bloch, Nitric oxide inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups, Circ. Res. 87 (2000) 140–145. Y. Luan, S. Chao, Z.Y. Ju, J. Wang, X. Xue, T.G. Qi, et al., Therapeutic effects of baicalin on monocrotaline-induced pulmonary arterial hypertension by inhibiting inflammatory response, Int. Immunopharmacol. 26 (2015) 188–193. W. Lee, S.K. Ku, S.W. Kim, J.S. Bae, Endocan elicits severe vascular inflammatory responses in vitro and in vivo, J. Cell. Physiol. 229 (2014) 620–630. J. Lu, H. Shimpo, A. Shimamoto, A.J. Chong, C.R. Hampton, D.J. Spring, et al., Specific inhibition of p38 mitogen-activated protein kinase with FR167653 attenuates vascular proliferation in monocrotaline-induced pulmonary hypertension in rats, J. Thorac. Cardiovasc. Surg. 128 (2004) 850–859. Z. Zeng, Y. Li, Z. Jiang, C. Wang, B. Li, W. Jiang, The extracellular signal-regulated kinase is involved in the effects of sildenafil on pulmonary vascular remodeling, Cardiovasc. Ther. 28 (2010) 23–29. L. Zuo, M. Lu, Q. Zhou, W. Wei, Y. Wang, Butyrate suppresses proliferation and migration of RKO colon cancer cells though regulating endocan expression by MAPK signaling pathway, Food Chem. Toxicol. 62 (2013) 892–900.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Inhibition of endocan attenuates monocrotaline-induced connective tissue disease related pulmonary arterial hypertension Haiyan Zhao a, Yunxin Xue b, Yun Guo a, Yue Sun a, Dongmei Liu a, Xiaofei Wang a,⁎ a b
Department of Immunology and Rheumatology, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China Department of Respiration, Liaoning Jinqiu Hospital, Shenyang 110016, People's Republic of China
a r t i c l e
i n f o
Article history: Received 11 July 2016 Received in revised form 31 October 2016 Accepted 18 November 2016 Available online xxxx Keywords: Endocan Pulmonary arterial hypertension Endothelial cells TNF-α MAPK
a b s t r a c t Connective tissue disease related pulmonary arterial hypertension (CTD-PAH) is characterized by vascular remodeling, endothelial dysfunction and inflammation. Endocan is a novel endothelial dysfunction marker. The aim of the present study was to investigate the role of endocan in CTD-PAH. Monocrotaline (MCT)-induced PAH rats were used as the CTD-PAH model. Short hairpin RNA packed in a lentiviral vector used to inhibit endocan expression was intratracheally instilled in rats prior to the MCT injection. Endocan was found to be increased in the serum and lung of MCT-induced PAH rats. Short hairpin RNA mediated knockdown of endocan significantly decreased right ventricular systolic pressure, attenuated pulmonary remodeling and inflammatory responses in the lung. In the in vitro study, tumor necrosis factor-α (TNF-α) exposure caused increased endocan expression in the primary cultured rat pulmonary microvascular endothelial cells (RPMECs). Endocan knockdown inhibited the permeability increase and adhesion molecules secretion in RPMECs induced by TNF-α. In addition, TNF-α induced MAPK activation was blocked when endocan gene was knocked down. These data demonstrate that endocan may play an important role in the development of CTD-PAH. This study provides novel evidence to better understand the pathogenesis of CTD-PAH, which may be beneficial for the treatment of this disease. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Pulmonary arterial hypertension (PAH) is a syndrome characterized by increased mean pulmonary arterial pressure (mPAP). PAH develops pulmonary vessel remodeling and changes the features of hemodynamic, and finally leads to right heart failure. Connective tissue disease (CTD) is closely associated with PAH. Patients with CTD are at increased risk for developing PAH [1]. The prevalence of PAH in systemic sclerosis, a CTD that is most commonly associated with PAH, is 7.85–13% of patients [2–4]. Moreover, CTD-related PAH (CTD-PAH) has a poor prognosis. In China, the survival rates of patients with CTD-PAH are inferior to those of patients with idiopathic PAH [5]. However, the pathogenesis of CTD-PAH has not been fully revealed yet. To investigate the possible molecular mechanisms of CTD-PAH would be beneficial for the treatment of this disease. Endothelial cell (EC) dysfunction is one of the pathological processes that contribute to the development of PAH. ECs are essential for vascular structure and functions. For example, ECs produce a number of cytokines that regulate the physical functions of pulmonary vessels, such ⁎ Corresponding author at: Department of Immunology and Rheumatology, Shengjing Hospital of China Medical University, No. 36 Sanhao Street, Heping District, Shenyang, Liaoning 110004, People's Republic of China. E-mail address: [email protected] (X. Wang).
http://dx.doi.org/10.1016/j.intimp.2016.11.016 1567-5769/© 2016 Elsevier B.V. All rights reserved.
as angiogenesis, vasoconstriction and vasodilatation. EC dysfunction results in imbalance of these cytokines, induces vasoconstriction and smooth muscle cells hypertrophy, and finally leads to vascular remodeling [6]. ECs also protect the smooth muscle cells and fibroblasts of the vessel from exposing to injurious factors. Damage of EC barrier integrity leads to vascular tissue cells expose to the excessive cytokines induced by stimulus and facilitates the injury. Increasing evidence has accumulated indicating that inflammation plays an integral role in the pathogenesis of PAH [7]. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine secreted by macrophages in response to inflammatory stimuli. Mice with overexpression of TNFα showed right ventricle hypertrophy and pulmonary hypertension [8], and TNF-α antagonist etanercept attenuate monocrotaline (MCT)-induced PAH [9,10]. In addition, TNF-α also plays a key role in the progress of CTD. TNF-α antagonists are widely used in patients with various CTD such as arthritis, systemic sclerosis, and ankylosing spondylitis [11–14]. Endocan, previously known as endothelial cell specific molecule-1 (ESM-1), is a soluble dermatan sulfate proteoglycan which was first cloned from human umbilical vein endothelial cell (HUVEC) cDNA library in 1996 [15]. Endocan serves as a regulator of vascular cell processes such as proliferation, adhesion and migration [15,16]. Endocan is considered as a novel EC dysfunction marker because it is mainly secreted by EC and its alteration often occurs in conditions characterized
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by endothelial dysfunction. Till now, dysregulation of endocan has been found in cancers [17,18], sepsis [19], cardiovascular diseases [20,21] and diabetic retinopathy [22]. However, at best of our knowledge, no study has reported the role of endocan in CTD-PAH. In the present study, virus packed endocan- short hairpin RNA (shRNA) was injected into MCT-induced rats, a CTD-PAH model, to investigate the role of endocan in this disease. In addition, TNF-α-induced primary pulmonary microvascular endothelial cells were also used to investigate possible underlying mechanisms in vitro. 2. Materials and methods
2.4. ELISA Lung tissue and primary endothelial cells were homogenized in cool PBS and repeated freezing in liquid nitrogen and thawing. The homogenate was then centrifuged at 12,000 g for 10 min at 4 °C. Protein concentrations in the supernatants were determined using a BCA protein assay kit (Beyotime). The levels of endocan and TNF-α in the serum, lung tissue or cell culture medium were analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Boster, Wuhan, China) following the manufactures' instructions.
2.1. Animals 2.5. Isolation and culture of RPMECs All animal studies were approved by the ethics committee of the China Medical University. Male Sprague-Dawley (SD) rats weighing 180–220 g were obtained from Laboratory Animal Center of China Medical University, (Shenyang, China). The rats were maintained in a temperature and humidity-controlled room (21– 22 °C, 75– 80%) with a 12/12-h light/dark cycle and had free access to standard rat chow and water ad libitum. The animals were randomly divided into: 1) control group (Con, n = 10); 2) MCT-induced pulmonary arterial hypertension group (PAH, n = 10); 3) PAH and scramble shRNA group (PAH + scr shRNA, n = 10); and 4) PAH and endocan shRNA group (PAH + endo shRNA, n = 10). The EGFP-encoding lentiviral strain carrying the shRNA oligonucleotides that target 5′-GGTGACGAGTTTGGTGTC-3′ on endocan mRNA or a scramble shRNA with the sequence of 5′TTCTCCGAACGTGTCACGT-3′ were obtained from Hanbio Co., Ltd. (Shanghai, China). Endocan shRNA or scramble shRNA was were administrated to the rats in the shRNA groups by intratracheal instillation through the mouth daily for 6 days (in total 1.5 × 108 transducing units in 300 μl PBS, 50 μl per day). Rats in the PAH groups received a single subcutaneous injection of 60 mg/kg MCT (Meilun, Dalian, China) which was dissolved in a mixed solution of ethanol and saline with the volume ratio of 2:8, at day 7. Rats in the control group received the same volume of vehicle. The rats were maintained for another 21 days. The blood was collected at day 1, 7, 14 and 21 after MCT injection.
Rat pulmonary microvascular endothelial cells (RPMECs) were isolated from pulmonary arteries as previously described [23]. Briefly, male SD rats (300–350 g) were anesthetized by an intraperitoneal injection of pentobarbital sodium. After thoracotomy, the pulmonary vasculature was perfused by injection of ice-cold PBS into the right ventricle. Lungs were then removed and placed in ice-cold serum-free Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA, USA). Thin strips were removed from the outermost surface of the lung periphery, minced and digested using 3% type II collagenase (Sigma-Aldrich, St Louis, MO, USA). The mixture was filtered through a 100 μm mesh, centrifuged at 300 ×g, and washed twice with cold PBS. The cells were resuspended in RPMI 1640 supplemented with 10% FBS (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin.
2.6. Inhibiting endocan expression in vitro Primary cultured RPMECs were infected with either Lenti-EGFPendocan-shRNA or Lenti-EGFP-scramble shRNA for 12, 24 and 48 h. The infection efficiency was examined by real time PCR and Western blotting.
2.7. Permeability assay in vitro 2.2. Right ventricular systolic pressure (RVSP) measurement At day 22, the rats were anesthetized intraperitoneally with 50 mg/kg of pentobarbital (i.p.). A heparin (0.3%) filled polyethylene catheter was introduced into the right ventricle through the right jugular vein. The catheter was connected to a BL-420F biological data acquisition and analysis system (Chengdu Techman Software Co., Ltd., Chengdu, China) using a pressure transducer. The digitalized RVSP was recorded. After the hemodynamic measurement, the rats were euthanized and the blood and lung tissue were collected for further analysis.
Permeability of RPMECs was quantitated spectrophotometrically by measuring the flux of Evans blue-bound albumin across RPMEC monolayers. RPMEC layer were incubated with indicated concentration of endocan in transwell chamber for 6 h. The cells were then washed with PBS for 3 times. Subsequently, fresh culture medium was added to the lower chamber, and the medium contain 0.67 mg/ml Evens blue and 4% BSA was added in the upper chamber. The optical density at 630 nm was measured in the lower chamber after 10 min incubation.
2.8. RNA isolation and quantitative real-time PCR 2.3. Histological analysis For lentiviral transduction efficiency determination in the lung, the lungs were perfused with PBS and fixed in 4% paraformaldehyde for 2 h. Next, the fixed lung tissues were frozen and cut into 10-μm-thick sections. The nucleoli were stained using 4′, 6-diamidino-2phenylindole (DAPI) and the intensity of EGFP was observed under a fluorescence microscope (BX53, Olympus, Tokyo, Japan). For H&E staining, the lung tissues were fixed in 4% paraformaldehyde overnight and embedded in paraffin. The paraffin blocks were cut into 5-μm-thick sections and stained with hematoxylin and eosin (H&E, Solarbio Science & Technology, Co., Ltd., Beijing, China). The sections were examined under a light microscopy. The index of pulmonary arterial wall thickness was calculated as the following formula: (external diameter-internal diameter) / external diameter × 100%.
Total RNA was isolated from lung tissue or RPMECs using a RNAsimple Total RNA Kit (TIANGEN Biotech, Beijing, China) following the manufacture's protocol. Complementary DNA (cDNA) was generated from total RNA using oligo-dT and Super Moloney Murine Leukemia Virus Reverse Transcriptase (BioTeke, Beijing, China). Real-time PCR reactions were each performed in a total volume of 20 μl reaction mixture, containing 1 μl cDNA, 10 μl 2 × SYBR Green Master Mix (BioTeke, Beijing, China), and 0.5 μl of each primer on an Exicycler 96 fluorescence quantitative detector (Bioneer, Daejeon, Korea). The transcript number was calculated using a 2−ΔΔCt method with β-actin as an internal reference. The primer sequences were listed as following: endocan: forward: 3′-TTCGGTGACGAGTTTGGTG-5′, reverse: 3′-TGTTTGGGAGGCAGAGGT5′; β-actin: forward: 3′-GGAGATTACTGCCCTGGCTCCTAGC-5′, reverse: 3′-GGCCGGACTCATCGTACTCCTGCTT-5′.
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2.9. Western blotting analysis Lung tissue or RPMECs were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). Total protein was extracted and concentration was determined using a Bicinchoninic Acid Protein Assay Kit (Beyotime). Total protein was separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis and the target proteins were transfer to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C and incubated with goat anti-rabbit IgG-HRP (1:5000; Beyotime) at room temperature for 45 min. Immunoreactive bands were visualized using enhanced chemiluminecent (7 Sea Pharmtech, Shanghai, China) and exposed on films (Fuji Photo Film, Tokyo, Japan). The gray values of blots were analyzed using a Gel-Pro-Analyzer software (Media Cybernetics, Rockville, MD, USA). The primary antibodies used in this study were as following: endocan antibody (1:500, PAC463Ra01, USCN Life Science, Wuhan, China), VCAM1 antibody (1:400, BA3840, Boster), ICAM1 antibody (1:400, PB0054, Boster) , E-selecin antibody (1:400, BA0615, Boster), ERK antibody (1:500, bs-2637R, Bioss, Beijing, China), pERK antibody (1:500, bs-1522R, Bioss), JNK antibody (1:500, bs-10562R, Bioss), pJNK antibody (1:500, bs-1640R, Bioss), p38 antibody (1:500, bs0637R, Bioss), and p-p38 antibody (1:500, bs-5477R, Bioss). 2.10. Statistical analysis Data were presented as the mean ± standard deviation (SD). Statistical tests were performed using SPSS software (19.0, IBM, New York, NY, USA). Comparisons between mean values of multiple groups were performed using one-way analysis of variance (ANOVA), followed by Fisher's least significant difference (LSD) post hoc test for multiple comparisons. P b 0.05 indicates statistically significant difference. 3. Results 3.1. shRNA mediated endocan inhibition in vivo Rat pulmonary tissues were observed under a fluorescence microscope 21 days after intratracheal gene transfer with Lenti-EGFP. Strong green fluorescence was observed in the pulmonary arterioles of rats from Lenti-EGFP treated groups, while no fluorescence was found in
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those of rats treated with PBS (Fig. 1A). In addition, endocan mRNA and protein levels were dramatically decreased in the lung of rats in the endocan-shRNA treated group (Fig. 1B and C). The scrambleshRNA has no effect on endocan expression. 3.1. Endocan inhibition attenuated right ventricular dysfunction and pulmonary inflammation Endocan expression level was measured in the lungs of MCT-exposed rats. As shown in Fig. 2A, the level of endocan in the lung was increased with time after treatment of MCT. The mRNA expression level of endocan was also significantly increased in the lungs of MCT-exposted rats (Fig. 1B). MCT induced an obvious increase in RVSP (36.03 ± 7.54 vs. 18.91 ± 3.17 mmHg, PAH vs. normal, P b 0.01, Fig. 2B). Endocan-shRNA administration in PAH rats resulted in significant attenuation of RVSP (27.3 ± 7.29 mm Hg, P b 0.01 vs. PAH). In addition, MCT exposure resulted in inflammatory response in the lungs as evidenced by significantly increased level of TNF-α (Fig. 2C), and this was suppressed by the inhibition of endocan. 3.2. Endocan inhibition attenuated pulmonary vessel remodeling The remodeling of the pulmonary arterial wall was determined. The lung tissue sections were stained using H&E and the pulmonary arterial wall thickness was calculated. In agreement with a previous study [24], MCT resulted in a significant increase in the index of pulmonary arterial wall thickness (38.1 ± 8.8% vs. 16.1 ± 4.2% PAH vs. normal, P b 0.01), which indicated a severe pulmonary arterial wall remodeling (Fig. 3). After inhibition of endocan expression by RNA interference, the thickening of the arterial wall induced by MCT was attenuated (21.5 ± 5.6%, P b 0.01 vs. PAH). 3.3. Effects of endocan inhibition on TNF-α-exposed RPMECs Endocan-shRNA-mediated RNAi were tested in RPMECs in vitro. Endocan knockdown induced a time-dependent decrease in mRNA and protein expression of endocan (Fig. 4A and B). The asynchronous expression of mRNA and protein may because the protein expression was later. Endocan-shRNA treated for 48 h was used in the following studies. TNF-α plays a key role in the progression of CTDs, including CTD-associated PAH [9]. Therefore, TNF-α-exposed RPMECs were used as an in
Fig. 1. Inhibition of endocan expression in vivo. Rat pulmonary tissues were observed under a fluorescence microscope after intratracheal gene transfer with Lenti-EGFP or PBS. Green fluorescence was found in the lung tissues of rats received Lenti6.3-EGFP-endocan-shRNA or Lenti-EGFP intratracheally but not in the rats received PBS (A). Results of quantitative real-time PCR (B) and western blot analysis (C) showed the decreased expression of endocan in the pulmonary tissues of endocan-shRNA treated rats. Values were represented as means ± SD (n = 5 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the PAH group. 1: Normal; 2: PAH; 3: PAH + endocan-shRNA; 4: PAH + scr-shRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Effects of endocan inhibition on PAH. Serum endocan was increased in MCT-induced PAH rats (A). Values were represented as means ± SD (n = 10 in each group). **P b 0.01 compared with the normal group at the same day; #P b 0.05 compared with the PAH group at day 1; ##P b 0.01 compared with the PAH group at day 1. Endocan knockdown decreased right ventricular systolic pressure (B) and reduced tumor necrosis factor-α level in the lung (C). Values were represented as means ± SD (n = 10 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the PAH group.
vitro model of CTD-associated PAH. As shown in Fig. 4C and D, TNF-α 20 ng/ml induced a markedly increase of endocan mRNA and protein expression in cultured RPMECs, which was consistent with the previous study [25]. To investigate the effect of endocan in TNF-α-exposed RPMECs, endocan expression was inhibited using endocan-shRNA. The decreased mRNA and protein expression of endocan indicated the success of the RNA interference. As illustrated in Fig. 4E and F, endocan administration induced a dose-dependent increase in permeability of RPMECs. In addition, endocan knockdown significantly inhibited TNF-α-induced permeability increase in RPMECs. 3.4. Effects of endocan on adhesion molecules expression in RPMECs The expression of the adhesion molecules on ECs is an important event in vascular inflammatory responses. In the present study, the expression levels of VCAM-1, ICAM-1 and E-selectin were detected in endocan-exposed RPMECs. As presented in Fig. 5A–D, endocan resulted in dose-dependently increases in the protein expression levels of VCAM-1, ICAM-1 and E-selectin. In addition, TNF-α also induced marked increases of the protein expression levels of VCAM-1, ICAM-1 and Eselectin in endothelial cell (Fig. 5E–H). However, shRNA-mediated endocan inhibition completely blocked the effects of TNF-α on the expression of these adhesion molecules, which indicated TNF-α may be involved in endocan-induced adhesion molecules expression in RPMECs. 3.5. Effects of endocan on the MAPK signaling pathway in RPMECs The MAPK signaling pathway is essential in TNF-α-induced ECs [26, 27], and also plays important roles in the pulmonary arterial ECs of MCT-induced PAH rats [28]. In this study, increased phosphorylation
levels of ERK, JNK and p38 was found in endocan-exposed RPMECs (Fig. 6A–C). TNF-α also caused significant increases in the phosphorylation levels of the MAPK proteins (Fig. 6D–F). Inhibiting endocan by shRNA completely prevented TNF-α-induced MAPK phosphorylation. 4. Discussion Endocan has been demonstrated to be abnormally expressed in various diseases, including cancer, cardiovascular diseases, and diabetes [17–22]. As an endothelial cell marker, endocan was reported to be involved in endothelial dysfunction and angiogenesis in these diseases. CTD-PAH is characterized by endothelial dysfunction and pulmonary arterial remodeling. Thus, the present study investigated the role of endocan in a rat model of CTD-PAH. The results demonstrated that endocan expression and serum endocan levels were increased in MCT-induced CTD-PAH rats. Short hairpin RNA-mediated endocan inhibition attenuated syndrome of CTD-PAH in the rats. In addition, TNF-α also resulted in an increase of endocan expression in the cultured RPMECs in vitro, and endocan knockdown inhibited the increase of endothelial permeability and adhesion molecules expression induced by TNF-α. Moreover, the MAPK signaling pathway may be involved in the pathological mechanisms of endocan. Among the animal models that have been developed for the study of PAH pathogenesis, MCT-induced PAH rat model was believed to be the model that could better mimic the progression and the inflammatory environment of CTD-PAH [9,29]. In the present study, the rats exposure to MCT showed significantly increased RVSP and TNF-α level in the lung, and obvious pulmonary arterial remodeling, which was consistent with the previous studies [30,31]. In addition, we found, for the first time, the serum level of endocan in MCT-induced PAH rats were increased. Considering the important role of endocan in endothelial functions, it is possibly involved in the endothelial dysfunction of CTD-PAH.
Fig. 3. Effects of endocan inhibition on pulmonary arterial remodeling in PAH rats. Lung tissue sections were stained using hematoxylin and eosin. Compared to tissue from normal rats (A), PAH rats showed thickened arterial wall (B), and shRNA-mediated endocan knockdown reversed this change (C). Scramble shRNA showed no effect on pulmonary arterial wall of PAH rats. The index of wall thickness was calculated and showed in (E). Scale bar: 100 μM. Values were represented as means ± SD (n = 10 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the PAH group.
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Fig. 4. Effects of endocan inhibition on TNF-α-exposed RPMECs. shRNA-meidated endocan RNAi time-dependently diminished endocan mRNA (A) and protein (B) expression in PRMECs. **P b 0.01 compared with the scr-shRNA group in the same time. TNF-α upregulated the mRNA (C) and protein (D) expression of endocan in RPMECs, and this was inhibited by endocan knockdown. Endocan induced a dose-dependent increase of permeability in RPMECs (E). Endocan knockdown inhibited TNF-α-induced increase of permeability in RPMECs (F). Values were represented as means ± SD (n = 5 in each group). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the TNF-α group. Numbers in panel B: 1, normal cells; 2, 4, 6, endo-shRNA 12, 24 and 48 h; 3, 5, 7, scr-shRNA 12, 24, and 48 h. Numbers in panel D: 1, normal cells; 2, TNF-α; 3, TNF-α + endo-shRNA; 4, TNF-α + scr-shRNA.
Moreover, shRNA mediated endocan gene knockdown decreased RVSP and pulmonary arterial remodeling, which confirmed the roles of endocan played in CTD-PAH.
In MCT-induced PAH model, inflammatory cells infiltrate into the lung tissues and secrete various inflammatory cytokines including TNF-α [32]. TNF-α also plays an important role in CTD. In the present
Fig. 5. Effects of endocan on the secretion of adhesion molecules in RPMECs. Endocan dose-dependently increased the protein expression of VCAM-1 (B), ICAM-1 (C) and E-selectin (D). Endocan knockdown inhibited the increased VCAM-1 (F), ICAM-1 (G) and E-selectin (H) induced by TNF-α. The reprehensive protein blots were shown in A and E. Values were represented as means ± SD (n = 3). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the TNF-α group.
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Fig. 6. Effects of endocan on the MAPK signaling pathway in RPMECs. Endocan induced phosphorylation of ERK (A), JNK (B) and p38 (C) in PRMECs. Endocan knockdown inhibited TNF-αinduced activation of MAPK proteins (D–F). Values were represented as means ± SD (n = 3). **P b 0.01 compared with the normal group; ##P b 0.01 compared with the TNF-α group. 1: Normal; 2: TNF-α; 3: TNF-α + endocan-shRNA; 4: TNF-α + scr-shRNA.
study, MCT-induced CTD-PAH rats showed increased TNF-α expression in the lung, and endocan knockdown reduced this increase. In the in vitro study, TNF-α induced upregulation of endocan expression in primary RPMECs, and endocan knockdown attenuated TNF-α-induced injury in the RPMECs. These results indicate that in the CTD-PAH there may be a reciprocal mechanism between endocan and TNF-α. These two cytokines can stimulate the expression of each other, and both of them contribute to the endothelial dysfunction. Therefore, endocan knockdown decreased the expression endocan itself and reduced the increased TNF-α induced by endocan, which attenuated endothelial dysfunction, and finally improved symptoms of PAH. As discussed above, inflammatory processes is the common characteristic of PAH and CTD. In addition to inflammatory cytokines like TNFα, vascular cell adhesion molecules also play important roles in inflammatory processes. Endothelial injury induces secretion of adhesion molecules and the excessive adhesion molecules cause further endothelial damage by facilitating leukocyte adhesion and migration to endothelium [33,34]. In the present study, the expression of VCAM-1, ICAM-1, and Eselectin was investigated. These adhesion molecules have been found to
be increased in CTD-PAH patients [35] and MCT-induced PAH models [36,37]. In addition, endocan upregulates the expression of these three cell adhesion molecules in ECs [38]. Our results demonstrated that endocan dose-dependently increased the expression of VCAM-1, ICAM1, and E-selectin in primary RPMECs, and endocan knockdown inhibited the expression of the three adhesion molecules induced by TNF-α in vitro. These results suggest that the expression of cell adhesion molecules regulated by endocan may be involved in the pathological mechanisms of CTD-PAH. The MAPK family includes signaling molecules that regulate various physiological and pathological processes, including cellular proliferation, differentiation, inflammatory responses and apoptosis. In response to stimuli, these signaling molecules are phosphorylated and further activate target genes or proteins. MAPK signaling has been found to be related with pathological processes in PAH such as pulmonary vascular remodeling and inflammatory responses [39,40]. Treatment with p38 MAPK inhibitor lowered the mean pulmonary artery pressure and reduced right ventricular hypertrophy in MCT-induced PAH rats [39]. Considering TNF-α exposure can induce MAPK phosphorylation, excessive
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TNF-α may contribute to the activation of MAPK in CTD-PAH. In the present study, endocan resulted in phosphorylation of ERK, JNK and p38, the three primary proteins in the MAPK family, and endocan knockdown completely blocked the effects of TNF-α on MAPKs. This result indicate that TNF-α activates MAPK through regulating endocan expression. In a previous study on RKO colon cancer cells [41], endocan expression was significantly increased in ERK2 knockdown cells. In our study, the reverse effect of MAPK on endocan has not been investigated. Whether there is feedback mechanism between MAPK and endocan in CTD-PAH need to be further studied. In summary, our study demonstrated that levels of endocan in the serum and lung were increased in MCT-induced CTD-PAH rats, and endocan knockdown by shRNA effectively attenuated PAH symptoms and reduced inflammatory responses in the lung. These results suggest that endocan may play an important role in the development of CTDPAH. This study provides novel evidence to better understand the pathogenesis of CTD-PAH, which may be beneficial for the treatment of this disease. Acknowledgements This study was supported by a grant from the Diagnostic and Treatment Capability Construction Project of Key Clinical Specialty for the Reform Programme of Liaoning Provincial Hospital (No. LNCCC-D 37-2015).
[17]
[18]
[19] [20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
References [1] Y.K. Sung, L. Chung, Connective tissue disease-associated pulmonary arterial hypertension, Rheum. Dis. Clin. N. Am. 41 (2015) 295–313. [2] D. Mukerjee, D. St George, B. Coleiro, C. Knight, C.P. Denton, J. Davar, et al., Prevalence and outcome in systemic sclerosis associated pulmonary arterial hypertension: application of a registry approach, Ann. Rheum. Dis. 62 (2003) 1088–1093. [3] E. Hachulla, V. Gressin, L. Guillevin, P. Carpentier, E. Diot, J. Sibilia, et al., Early detection of pulmonary arterial hypertension in systemic sclerosis: a French nationwide prospective multicenter study, Arthritis Rheum. 52 (2005) 3792–3800. [4] S. Phung, G. Strange, L.P. Chung, J. Leong, B. Dalton, J. Roddy, et al., Prevalence of pulmonary arterial hypertension in an Australian scleroderma population: screening allows for earlier diagnosis, Intern. Med. J. 39 (2009) 682–691. [5] R. Zhang, L.Z. Dai, W.P. Xie, Z.X. Yu, B.X. Wu, L. Pan, et al., Survival of Chinese patients with pulmonary arterial hypertension in the modern treatment era, Chest 140 (2011) 301–309. [6] M. Mandegar, Y.C. Fung, W. Huang, C.V. Remillard, L.J. Rubin, J.X. Yuan, Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension, Microvasc. Res. 68 (2004) 75–103. [7] M. Humbert, N.W. Morrell, S.L. Archer, K.R. Stenmark, M.R. MacLean, I.M. Lang, et al., Cellular and molecular pathobiology of pulmonary arterial hypertension, J. Am. Coll. Cardiol. 43 (2004) 13S–24S. [8] M. Fujita, J.M. Shannon, C.G. Irvin, K.A. Fagan, C. Cool, A. Augustin, et al., Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension, Am. J. Physiol. Lung Cell. Mol. Physiol. 280 (2001) L39–L49. [9] L.L. Zhang, J. Lu, M.T. Li, Q. Wang, X.F. Zeng, Preventive and remedial application of etanercept attenuate monocrotaline-induced pulmonary arterial hypertension, Int. J. Rheum. Dis. 19 (2016) 192–198. [10] Q. Wang, X.R. Zuo, Y.Y. Wang, W.P. Xie, H. Wang, M. Zhang, Monocrotaline-induced pulmonary arterial hypertension is attenuated by TNF-alpha antagonists via the suppression of TNF-alpha expression and NF-kappaB pathway in rats, Vasc. Pharmacol. 58 (2013) 71–77. [11] T. Cobo-Ibanez, M.A. Descalzo, E. Loza-Santamaria, L. Carmona, S. Munoz-Fernandez, Serious infections in patients with rheumatoid arthritis and other immune-mediated connective tissue diseases exposed to anti-TNF or rituximab: data from the Spanish registry BIOBADASER 2.0, Rheumatol. Int. 34 (2014) 953–961. [12] A. Saraux, J. Benichou, L. Guillevin, L. Idbrik, C. Job-Deslandre, J. Sibilia, et al., Which patients with rheumatoid arthritis, spondyloarthritis, or juvenile idiopathic arthritis receive TNF-alpha antagonists in France? The CORPUS cohort study, Clin. Exp. Rheumatol. 33 (2015) 602–610. [13] G. Murdaca, F. Spano, M. Contatore, A. Guastalla, F. Puppo, Potential use of TNF-alpha inhibitors in systemic sclerosis, Immunotherapy 6 (2014) 283–289. [14] L.J. Maxwell, J. Zochling, A. Boonen, J.A. Singh, M.M. Veras, E. Tanjong Ghogomu, et al., TNF-alpha inhibitors for ankylosing spondylitis, Cochrane Database of Syst. Rev. 4 (2015), CD005468. [15] P. Lassalle, S. Molet, A. Janin, J.V. Heyden, J. Tavernier, W. Fiers, et al., ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines, J. Biol. Chem. 271 (1996) 20458–20464. [16] D. Bechard, A. Scherpereel, H. Hammad, T. Gentina, A. Tsicopoulos, M. Aumercier, et al., Human endothelial-cell specific molecule-1 binds directly to the integrin
[28]
[29]
[30]
[31]
[32]
[33]
[34] [35]
[36]
[37]
[38] [39]
[40]
[41]
121
CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1, J. Immunol. 167 (2001) 3099–3106. N. Liu, L.H. Zhang, H. Du, Y. Hu, G.G. Zhang, X.H. Wang, et al., Overexpression of endothelial cell specific molecule-1 (ESM-1) in gastric cancer, Ann. Surg. Oncol. 17 (2010) 2628–2639. L.Y. Chen, X. Liu, S.L. Wang, C.Y. Qin, Over-expression of the endocan gene in endothelial cells from hepatocellular carcinoma is associated with angiogenesis and tumour invasion, J. Int. Med. Res. 38 (2010) 498–510. C. Palmiere, M. Augsburger, Endocan measurement for the postmortem diagnosis of sepsis, Legal Med. 16 (2014) 1–7. S. Balta, D.P. Mikhailidis, S. Demirkol, C. Ozturk, E. Kurtoglu, M. Demir, et al., Endocan–a novel inflammatory indicator in newly diagnosed patients with hypertension: a pilot study, Angiology 65 (2014) 773–777. X.S. Wang, W. Yang, T. Luo, J.M. Wang, Y.Y. Jing, Serum endocan levels are correlated with the presence and severity of coronary artery disease in patients with hypertension, Genet. Test. Mol. Biomarkers 19 (2015) 124–127. A.M. Abu El-Asrar, M.I. Nawaz, G. De Hertogh, A.S. Al-Kharashi, K. Van den Eynde, G. Mohammad, et al., The angiogenic biomarker endocan is upregulated in proliferative diabetic retinopathy and correlates with vascular endothelial growth factor, Curr. Eye Res. 40 (2015) 321–331. J. King, T. Hamil, J. Creighton, S. Wu, P. Bhat, F. McDonald, et al., Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes, Microvasc. Res. 67 (2004) 139–151. L.Q. Bi, R. Zhu, H. Kong, S.L. Wu, N. Li, X.R. Zuo, et al., Ruscogenin attenuates monocrotaline-induced pulmonary hypertension in rats, Int. Immunopharmacol. 16 (2013) 7–16. D. Bechard, V. Meignin, A. Scherpereel, S. Oudin, G. Kervoaze, P. Bertheau, et al., Characterization of the secreted form of endothelial-cell-specific molecule 1 by specific monoclonal antibodies, J. Vasc. Res. 37 (2000) 417–425. H. Choi, H.N. Nguyen, F.S. Lamb, Inhibition of endocytosis exacerbates TNF-alpha-induced endothelial dysfunction via enhanced JNK and p38 activation, Am. J. Physiol. Heart Circ. Physiol. 306 (2014) H1154–H1163. W. Olejarz, D. Bryk, D. Zapolska-Downar, M. Malecki, A. Stachurska, D. Sitkiewicz, Mycophenolic acid attenuates the tumour necrosis factor-alpha-mediated proinflammatory response in endothelial cells by blocking the MAPK/NF-kappaB and ROS pathways, Eur. J. Clin. Investig. 44 (2014) 54–64. A.C. Church, D.H. Martin, R. Wadsworth, G. Bryson, A.J. Fisher, D.J. Welsh, et al., The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension, Am. J. Physiol. Lung Cell. Mol. Physiol. 309 (2015) L333–L347. D.W. Wilson, H.J. Segall, L.C. Pan, S.K. Dunston, Progressive inflammatory and structural changes in the pulmonary vasculature of monocrotaline-treated rats, Microvasc. Res. 38 (1989) 57–80. A. Csiszar, N. Labinskyy, S. Olson, J.T. Pinto, S. Gupte, J.M. Wu, et al., Resveratrol prevents monocrotaline-induced pulmonary hypertension in rats, Hypertension 54 (2009) 668–675. Y. Sadamura-Takenaka, T. Ito, S. Noma, Y. Oyama, S. Yamada, K. Kawahara, et al., HMGB1 promotes the development of pulmonary arterial hypertension in rats, PLoS One 9 (2014), e102482. T.M. Kolb, R.L. Damico, P.M. Hassoun, Linking new and old concepts: inflammation meets the Warburg phenomenon in pulmonary arterial hypertension, J. Mol. Med. 89 (2011) 729–732. C.J. Kirkpatrick, M. Wagner, I. Hermanns, C.L. Klein, H. Kohler, M. Otto, et al., Physiology and cell biology of the endothelium: a dynamic interface for cell communication, Int. J. Microcirc. Clin. Exp. 17 (1997) 231–240. J. Panes, M. Perry, D.N. Granger, Leukocyte-endothelial cell adhesion: avenues for therapeutic intervention, Br. J. Pharmacol. 126 (1999) 537–550. F. Iannone, M.T. Riccardi, S. Guiducci, R. Bizzoca, M. Cinelli, M. Matucci-Cerinic, et al., Bosentan regulates the expression of adhesion molecules on circulating T cells and serum soluble adhesion molecules in systemic sclerosis-associated pulmonary arterial hypertension, Ann. Rheum. Dis. 67 (2008) 1121–1126. J.D. Roberts Jr., J.D. Chiche, J. Weimann, W. Steudel, W.M. Zapol, K.D. Bloch, Nitric oxide inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups, Circ. Res. 87 (2000) 140–145. Y. Luan, S. Chao, Z.Y. Ju, J. Wang, X. Xue, T.G. Qi, et al., Therapeutic effects of baicalin on monocrotaline-induced pulmonary arterial hypertension by inhibiting inflammatory response, Int. Immunopharmacol. 26 (2015) 188–193. W. Lee, S.K. Ku, S.W. Kim, J.S. Bae, Endocan elicits severe vascular inflammatory responses in vitro and in vivo, J. Cell. Physiol. 229 (2014) 620–630. J. Lu, H. Shimpo, A. Shimamoto, A.J. Chong, C.R. Hampton, D.J. Spring, et al., Specific inhibition of p38 mitogen-activated protein kinase with FR167653 attenuates vascular proliferation in monocrotaline-induced pulmonary hypertension in rats, J. Thorac. Cardiovasc. Surg. 128 (2004) 850–859. Z. Zeng, Y. Li, Z. Jiang, C. Wang, B. Li, W. Jiang, The extracellular signal-regulated kinase is involved in the effects of sildenafil on pulmonary vascular remodeling, Cardiovasc. Ther. 28 (2010) 23–29. L. Zuo, M. Lu, Q. Zhou, W. Wei, Y. Wang, Butyrate suppresses proliferation and migration of RKO colon cancer cells though regulating endocan expression by MAPK signaling pathway, Food Chem. Toxicol. 62 (2013) 892–900.