Exposure to endosulfan increases endothelial permeability by transcellular and paracellular pathways in relation to cardiovascular diseases

Environmental Pollution xxx (2017) 1e9

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Exposure to endosulfan increases endothelial permeability by transcellular and paracellular pathways in relation to cardiovascular diseases* Dan Xu, Tong Liu, Limei Lin, Shuai Li, Xiaoming Hang, Yeqing Sun* Institute of Environmental Systems Biology, Dalian Maritime University, Linghai Road 1, Dalian, 116026, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 August 2016 Received in revised form 19 November 2016 Accepted 20 December 2016 Available online xxx

Exposure to environmental pollutants results in out-of-balance of vascular homeostasis. Endothelial dysfunction leads to a disruption of the endothelial permeability characteristics, associated with cardiovascular diseases. We previously reported that endosulfan could cause endothelial dysfunction, but the role of endosulfan in permeability of endothelial cells has been unexplored. To elucidate molecular mechanism of endosulfan-induced changes in endothelial permeability, human umbilical vein endothelial cells (HUVECs) were exposed to endosulfan, followed by endothelial permeability analysis. The results showed that permeability of HUVECs was enhanced at 48 h after exposure to endosulfan in a dose-dependent manner. Immunofluorescence analysis demonstrated the disruptions of actin cytoskeleton and focal adhesion in endosulfan-exposed cells. Endosulfan activated MMP3/LAMC1/FAK signaling pathway, and downregulated ROCK and PXN in transcellular pathway. Endosulfan affected adherens junctions via E-cadherin and b-catenin, and impaired gap junctions through downregulation of Cx43 in paracellular pathway. We predicted four closely related human cardiovascular diseases in Nextbio, including shock, coronary arteriosclerosis, disorder of cardiac function and hypertensive disorder in relation to endosulfan exposure. Some genes such as ROCK2 and PXN were predicted to be key genes in these diseases. These findings suggest that endosulfan increased endothelial permeability by paracellular and transcellular pathways, implicating the potential correlation between endosulfan and cardiovascular diseases. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Endosulfan Endothelial permeability Actin cytoskeleton Interendothelial junctions Cardiovascular diseases

The main finding of the present study is that endosulfan increases endothelial permeability by paracellular and transcellular pathways, resulting in the loss of barrier role of endothelial cells and thereby contributing to cardiovascular diseases. 1. Introduction Endosulfan is one of the organochlorine pesticides, which belongs to persistent organic pollutants (POPs) (Becker et al., 2011). It has been used in agriculture worldwide during the past 50 years (Gandhi et al., 2015), thereby has a widespread distribution in the environment and becomes a ubiquitous environment contaminant

*

This paper has been recommended for acceptance by Prof. von Hippel Frank A. * Corresponding author. E-mail addresses: [email protected] (D. Xu), [email protected] (T. Liu), [email protected] (L. Lin), [email protected] (S. Li), [email protected] (X. Hang), [email protected] (Y. Sun).

due to potential transport in India, China, and other countries (Song et al., 2012; Weber et al., 2010). Endosulfan is mainly absorbed into human body via the skin, lungs, stomach and even placenta (LopezEspinosa et al., 2008). It is reported that the milk concentration of endosulfan was 0.4e56.2 mg/ml, and human blood concentration of endosulfan was 0.69e176.2 mg/ml in Kerala Kasargod District of India (Singh et al., 2007) where endosulfan has been used for several decades. The influence of endosulfan exposure on human health has been of great concern today. Environmental risk assessment studies have demonstrated that exposure to endosulfan could result in acute, subacute and chronic risks in human health (Chauhan et al., 2016) due to its toxicity to different organ systems such as nervous system (Enhui et al., 2016; Skandrani et al., 2006), immune system (Kannan et al., 2000; Zhao et al., 2014), hepatic (Lu et al., 2000; Moses and Peter, 2010; Peyre et al., 2012) and cardiovascular systems (Li et al., 2015; Ozmen, 2013). Most of these studies are based on endosulfan-induced cytotoxicity by culturing some cell lines for

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Please cite this article in press as: Xu, D., et al., Exposure to endosulfan increases endothelial permeability by transcellular and paracellular pathways in relation to cardiovascular diseases, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2016.12.051

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several hours or days in the presence of increasing concentrations of endosulfan ranging from about 1 to 245 mM (1e100 mg/ml). IC50 (concentration leading to a 50% decrease of cell growth) is the important reference concentration in cytotoxicity studies. The IC50 value for endosulfan in human umbilical vein endothelial cells (HUVECs) was calculated as 62.8 mM using MTT assay in our previous study (Li et al., 2015). Accordingly, endosulfan at the concentrations of 20, 40 and 60 mM were chosen in the present study. Endosulfan at 20 mM significantly inhibited cell growth, so the concentration of 20 mM can be regarded as a start concentration of endosulfan cytotoxicity in human endothelial cells. Vascular endothelial cells are thought to be the natural physiological barrier between the blood and the vessel wall (Komarova and Malik, 2010). It is known that the regulation of endothelial permeability contributes to the maintenance of endothelial barrier function through transcellular and paracellular pathways. Transcellular pathway can regulate endothelial permeability via actin cytoskeleton. Previous study suggested that hemorrhagic shock resulted in the loss of this intestinal barrier through the disruption of filamentous actin (F-actin) in transcellular pathway (Thuijls et al., 2009). It is reported that the loss of F-actin in human microvascular endothelial cells exposed to perfluorooctane sulfonate (PFOS) played a critical role in endothelial permeability (Qian et al., 2010). Rho associated coiled-coil containing protein kinase-2 (ROCK2) and paxillin (PXN) are well-known cytoskeletal proteins. Loss of ROCK2 activity inhibited the formation of actin stress fibers in endothelial cells (Liu et al., 2013), associated with the pathogenesis of atherosclerosis and cardiovascular diseases (Mukai et al., 2001). PXN was involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix, implied in pulmonary hypertension (Veith et al., 2012). Focal adhesion kinase (FAK) is one of components of focal adhesions, localized at the ends of actin stress fibers (Zhang et al., 2014). Paracellular pathway is associated with endothelial permeability by the interendothelial junctions that consist of adherens and gap junctions. Adherens junctions (AJs) are cadherin/ catenin-containing adhesive structures, and E-cadherin is linked to the actin cytoskeleton by its direct interaction with b-catenin (Kam and Quaranta, 2009). Recent study shows that posthemorrhagic shock mesenteric lymph enhanced monolayer permeability of endothelial cells via F-actin and vascular endothelial (VE)-cadherin (Sun et al., 2016). Gap junctions (GJs) form channels between adjacent cells and transmit signals that modulate gene transcription, cytoskeleton and growth control within the contiguous monolayer of cells (Giepmans, 2004). In vertebrates, GJs are comprised of connexins (Cxs), a family of integral membrane proteins (Willecke et al., 2002). Connexin 43 (Cx43) has been identified in endothelial cells (Ho et al., 2013) and functions on gap junction intercellular communication (GJIC). Therefore, the integrity of actin cytoskeleton and interendothelial junctions are critical for the regulation of endothelial permeability. Cardiovascular diseases are the leading cause of death globally, including vascular diseases and heart diseases (Balfour et al., 2016; Bundy and He, 2016). Vascular endothelial cells play an important role in regulating cardiovascular homeostasis. The permeability of endothelial barrier is exquisitely regulated process in response to extracellular stimuli. HUVECs have considerably been used for the investigation of endothelial hyperpermeability (Ong et al., 2013; Sun et al., 2016; Yao et al., 2015). We previously reported that endosulfan induced endothelial dysfunction by inhibition of cell growth and induction of inflammation in HUVECs (Li et al., 2015). Gene expression profile analysis identified that low dose exposure of endosulfan was associated with the regulation of actin cytoskeleton (Xu et al., 2016) that governs permeability. In the present study, we observed the influence of endosulfan on the permeability of HUVECs and further analyzed the changes in transcellular and

paracellular pathways of endothelial permeability. Subsequently, we predicted closely related human cardiovascular diseases and key genes when exposure to endosulfan using the NextBio Human Disease Atlas. This study will be helpful for better understanding the correlation between endosulfan and cardiovascular diseases. 2. Materials and methods 2.1. Cell culture and endosulfan exposure HUVECs were obtained from Shanghai Institutes for Biological Sciences, China. Cells were maintained in RPMI-1640 (KeyGen, Nanjing, China) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37
C in a humidified incubator with 5% CO2. Endosulfan (Jiangsu Anpon Electrochemical Co, Huaian, China) was dissolved in dimethyl sulfoxide (DMSO, SigmaeAldrich, MO) to yield a 0.5 M stock solution. Endosulfan exposure experiments were performed as previously described (Li et al., 2015). The concentrations of endosulfan at 20 mM (equal to 8.16 mg/ml), 40 mM (equal to 16.33 mg/ml) and 60 mM (equal to 24.48 mg/ml) were within the detected concentration range of endosulfan (0.69e176.2 mg/ml) in human blood from people living in the endosulfan exposed area (Singh et al., 2007). 2.2. Endothelial permeability analysis Monolayer permeability was determined by using a Coaster transwell system (Corning Inc, Corning, NY). HUVECs were exposed to endosulfan or transfected with siRNAs in cell culture inserts that were mounted onto 6-well culture plates. After cells were treated for 48 h, cells reached confluence forming HUVECs monolayer in the upper chambers. The Texas Redelabelled dextran tracer (final concentration 20 mg/ml, Invitrogen) was added into each of the upper chambers and incubated at 37
C for another 2 h. The amount of tracer that penetrated through the endothelial monolayer into the lower chamber was measured by a Microplate System (SpectraMax M5, Molecular Devices, CA). The permeability index was determined by comparing the concentrations of the tracer between the upper chamber and the lower chamber (Yan et al., 2006). 2.3. Immunofluorescence analysis HUVECs were seeded in an 8-well chamber culture slides (BD Biosciences, San Jose, CA). Cells were fixed with 4% formaldehyde and permealized with 0.2% Triton-X 100. After blocking with 3% BSA, cells were incubated with anti-FAK antibody (1:200, Upstate), followed by goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500, Molecular Probes). F-actin was labelled with Rhodamine conjugated phalloidin (1:100, Molecular Probes). Cells were mounted in aqueous mounting medium containing DAPI (0.25 mg/ml). Images were acquired using 40  objective with laser confocal fluorescent microscopy (Lecia TCS SP5 II, Germany) and the average fluorescence intensity of F-actin from three randomly selected fields was monitored by quantify analysis. 2.4. Quantitative RT-PCR analysis Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis was performed with SYBR Green (Invitrogen) using an ABI PRISM 7300 system (Applied Biosystems, CA). qRT-PCR reactions were performed in triplicate from all the samples in different groups. The PCR primer sequences (50 -30 ) were designed for ACTB: F:50 -

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CCTGTACGCCAACACAGTGC-30 , R:50 -ATACTCCTGCTTGCTGATCC-3’; ROCK2: F:50 -GAAGTGGGTTAGTCGGTTGG-30 , R:50 -CAGTTAGCTAGGTTTGTTTGGG-3’; PXN: F:50 -GAGAAGCCTAAGCGGAATGG-30 , R:50 AGATGCGTGTCTGCTGTTGG-3’; MMP3: F:50 -ACAAGGAGGCAGGCAAGAC-30 , R:50 CACGCACAGCAACAGTAGGA-3’; Cx43: F:50 CTCGCCTATGTCTCCTCCTG-30 , R:50 GCTGGTCCACAATGGCTAGT-3’; GAPDH: F:50 -GGGAAACTGTGGCGTGAT-30 , R:50 GAGTGGGTGTCGCTGTTGA-3’. Relative expression levels of mRNAs among samples were calculated using the comparative delta CT method (2△△CT) after normalization with reference to the expression of GAPDH. 2.5. Western blot analysis Whole cell protein lysates were harvested by lysis buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 10 mM NaF, 0.2 mM Na3VO4 and protease inhibitor (SigmaeAldrich)]. Proteins (30 mg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA). Primary antibodies include MMP-1, 3 (R&D Systems, Shanghai, China), FAK (Upstate), p-FAK (Tyr 397) and LAMC1 (Santa Cruz, Delaware, CA), E-cadherin and b-catenin (BD Biosciences). GAPDH (ZSGQ-BIO, Beijing, China) was used as a loading control. The secondary antibodies were HRPconjugated anti-rabbit (NA 934 V) and anti-mouse (NA 931 V) antibodies (GE Healthcare Life Sciences, Wellesley, MA). Proteins were visualised by chemiluminescence according to the manufacturer's instructions (Thermo, Pittsburgh, PA), followed by exposure to Xray film. The density of bands was densitometrically quantified using Image J (National Institutes of Health, Bethesda, MD). 2.6. Transient siRNA transfection Negative control siRNA (NC siRNA) and LAMC1 siRNAs were purchased from QIAGEN (Valencia, CA) and Biomics (Nantong, China), respectively. Cells were transfected with 10 nM of siRNA using LipofectamineRNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Transfection efficiency we adopted in this study was estimated as >90% using a fluorescence-labelled double-stranded siRNA (Biomics). 2.7. Cell growth assay HUVECs were plated in 35 mm dishes and grown to 30%e50% confluence prior to siRNA transfection. Cells were collected and stained with 0.4% trypan blue at 48 h after siRNA transfection. Viable cells were counted using a hemocytometer under an inverted light microscope (Nikon ECLIPSE TS100). For each sample, the experiments were performed in triplicates (n ¼ 3). 2.8. Gap junction activity assay HUVECs were plated in 35 mm dishes and Lucifer yellow (LucY, 0.5 mg/ml) (Sigma-Aldrich) was added at the center of the dishes when cell were grown to confluency. Using a razor blade, three scrapes per dish were made to allow LucY dye entry and incubated in dark for 10 min. Then, cells were washed, fixed in 4% paraformaldehyde and photographed using 20  objective under an inverted fluorescent microscope (Nikon ECLIPSE TE2000-E). The degree of LucY dye transfer was quantified by measuring the maximal distances from the scrape lines to the travel front of LucY (Zhao et al., 2006). At least five images for each scrape wound per well were used to assess the level of dye transfer in three independent experiments.

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2.9. Disease prediction analysis Gene expression profiles from the low dose (20 mM) endosulfanexposed cells versus DMSO control were analyzed in NextBio (http://nextbio.com) to identify closely related human cardiovascular diseases as described in our previous study (Xu et al., 2016). Pairwise gene signature correlations and rank-based enrichment statistics were employed to calculate the NextBio scores for each disease. 2.10. Statistical analysis All data are presented as the mean ± SD from at least three independent experiments. Statistical comparisons between groups were performed with two-tailed t-tests using Microsoft Excel 2010. *P < 0.05, **P < 0.01. 3. Results 3.1. Changes in endothelial permeability and actin cytoskeleton in HUVECs To study the effect of endosulfan on monolayer endothelial permeability, we utilized a transwell system and a Texas Redelabelled Dextran tracer to detect the change in permeability of HUVECs. Results showed that endosulfan significantly enhanced endothelial permeability in a dose-dependent manner when HUVECs were exposed to endosulfan at 20, 40 and 60 mM dose for 48 h (P < 0.01, Fig. 1A). It is known that the cytoskeleton is vital to the maintenance of vascular permeability (Bogatcheva and Verin, 2008). In DMSO control group, most of HUVECs have normal structure of actin cytoskeleton. Actin stress fibers were clearly detected and mainly distributed in the cytoplasm while FAK localized at the ends of stress fibers. Endosulfan at 20 mM dose reduced F-actin staining and caused the loss of FAK localization at the ends of stress fibers (Fig. 1B) in HUVECs. The formation of stress fibers was inhibited by endosulfan, which was confirmed by quantify analysis of fluorescent intensity of stress fibers (Fig. 1C). As shown in Fig. 1D, qRT-PCR results confirmed that ROCK2 and PXN were significantly downregulated in endosulfan-exposed group in contrast to DMSO control group, supporting the results about disruptions of actin cytoskeleton and focal adhesion in HUVECs exposed to endosulfan. 3.2. Effects of endosulfan on MMP-3, LAMC1 and FAK expression Proteins of the matrix metalloproteinase (MMP) family including collagenases (MMP-1), gelatinases (MMP-2), stromelysins (MMP-3) and others are involved in the breakdown of extracellular matrix (ECM) in normal physiological processes (Newby, 2006). Our previous study from microarray (Xu et al., 2016) found that endosulfan remarkably increased mRNA expression of MMP-1 and MMP-3. qRT-PCR results confirmed that MMP-3 mRNA were elevated in all dose of endosulfan exposed groups. In this study, MMP-3 protein expression in 40 and 60 mM endosulfan-exposed groups was significantly higher than that of control (C) or DMSO (D) groups whereas MMP-1 protein expression almost had no obvious change in endosulfan-exposed groups (Fig. 2A). It is known that MMP-3 degrades fibronectin, laminin, gelatins, collagens and cartilage proteoglycans, involved in wound repair and progression of atherosclerosis (Kunz, 2007). Therefore, we suppose that endosulfan might affect the downstream substrates of MMP-3, such as laminin. We focus on LAMC1 (encoding laminin g-1), one of laminin family, because LAMC1 was implied in inflammation process and atherosclerosis (Rauch et al., 2011; Spenle et al., 2012). We also

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Fig. 1. Effects of endosulfan on endothelial permeability and actin cytoskeleton. (A) Endothelial permeability analysis. (B) F-actin was labelled with Rhodamine conjugated phalloidin in red. FAK was detected in green at the ends of stress fibers and in the cytoplasm. Scale bar, 25 mm. (C) Fluorescent intensity of stress fibers per cell was quantified from at least 60 cells in three random fields. (D) ROCK2 and PXN at mRNA levels were examined by qRT-PCR. **P < 0.01 vs DMSO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

found that LMAC1 was a putative target gene of hsa-miR-22 that was remarkably elevated in endosulfan-exposed HUVECs (unpublished data), indicating LAMC1 might be involved in endosulfaninduced endothelial dysfunction. In this study, endosulfan significantly suppressed the protein expression of LAMC1 (Fig. 2B), companied with the decrease in FAK and p-FAK (Tyr 397). These findings suggest that endosulfan increased MMP-3 expression to degrade LAMC1 and subsequently lead to FAK activity, suggesting that LAMC1 might be associated with the alteration of actin cytoskeleton and focal adhesion induced by endosulfan.

3.3. Involvement of LAMC1 in regulating endothelial function To study the effect of LAMC1 on endothelial function, we designed three siRNAs against LAMC1. HUVECs were not transfected and transfected with LAMC1 siRNAs (si-1, si-2, si-3) or NC siRNA (NC) for 48 h. The results from Western blot analysis (Fig. 3A) confirmed that LAMC1 was knocked down by each siRNA. LAMC1 siRNAs (si-1 and si-2) resulted in the decrease in FAK and p-FAK (Tyr 397), and significantly inhibited cell growth (Fig. 3B) in HUVECs. Further, we found that downregulation of LAMC1 inhibited stress fiber formation (data not shown) and significantly reduced mRNA expression of ROCK2 and PXN in HUVECs (Fig. 3C). LAMC1 siRNAs could significantly enhance endothelial

permeability (Fig. 3D). These findings suggest the involvement of LAMC1 in regulating endothelial barrier function.

3.4. Effects of endosulfan on adherens junctions and gap junctions To investigate the effect of endosulfan in paracellular pathway of endothelial permeability, we observed the changes in the interendothelial junctions including AJs and GJs when HUVECs were exposed to varying concentrations of endosulfan for 48 h. The results showed that E-cadherin and b-catenin in AJs were significantly downregulated in lower doses (20 and 40 mM) endosulfanexposed groups (Fig. 4A), but they appeared no obvious changes at 60 mM dose of endosulfan. To evaluate the degree of gap junctional communication, we used a scrape-loading paradigm and the GJ permeant dye LucY. Cells in DMSO group were extensively coupled, as determined by the spread of the fluorescent dye from the edge of the cut (Fig. 4B). Endosulfan exposure significantly reduced the distance of scrape, resulting in marked loss of dye coupling (Fig. 4C). Downregulation of Cx43 expression was confirmed by qRT-PCR analysis in endosulfan-exposed HUVECs (Fig. 4D), indicating that endosulfan inhibited gap junction activity in HUVECs.

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Fig. 2. Effects of endosulfan on MMP-3/LAMC1/FAK signaling pathway. (A) Expression of MMP-1 and MMP3 proteins. (B) Expression of LAMC1, FAK and p-FAK(Tyr397) proteins. Representative western blots were shown and protein levels were densitometrically quantified as the mean ± SD (n ¼ 3). *P < 0.05, **P < 0.01 vs DMSO.

3.5. Human cardiovascular disease prediction To reveal the potential correlation between endosulfan and cardiovascular diseases, we utilized differentially expressed genes to predict the closely related human cardiovascular diseases by NextBio analysis in 20 mM endosulfan-exposed groups (Table 1). Results showed that shock (score 71), coronary arteriosclerosis (score 55), disorder of cardiac function (score 55) and hypertensive disorder (score 46) were listed the top four cardiovascular diseases predicted to be associated with endosulfan exposure. Interestingly, several genes including SLC26A1, PXN and ROCK2 commonly altered in two of these cardiovascular diseases. Notably, we found that LAMA4 (encoding laminin a4), one of the laminin family, was predicted to be key downregulated gene in hypertensive disorder such as pulmonary arterial hypertension. It is reported that LAMA4 promoted migration, proliferation, and survival of endothelial cells (Shan et al., 2015). We suppose that all or part of these predicted key genes might be associated with cardiovascular diseases, which provides valuable and scientific basis for the potential correlation between endosulfan and cardiovascular diseases.

4. Discussion In the past several decades, the effects of environment toxicants on human health have been of great concern. Monolayer endothelial cells are a physiologic barrier to keep a balance of vascular homeostasis (Komarova and Malik, 2010). The increase of endothelial permeability is a prevalent feature among vascular pathologic processes due to endothelial dysfunction (Anderson, 1999). Currently, disruption of actin cytoskeleton and interendothelial junction are the most commonly accepted initiative factors of increased vascular permeability, resulting in the loss of barrier role

of endothelial cells. In the present study, we at the first time reported that endosulfan exposure may increase endothelial permeability in HUVECs through transcellular and paracellular pathways. We predicted four closely related human cardiovascular diseases and a number of key genes such as PXN and ROCK2, indicating the potential correlation between endosulfan and cardiovascular diseases. The Rho family of GTPases such as RhoA regulate stress fiber formation through its downstream ROCK2, which is responsible to the formation of stress fibers and the maintenance of the integrity of stress fibers. FAK and PXN are representative proteins in focal adhesions, connected with stress fibers. In the present study, endosulfan reduced the expression levels of ROCK2, PXN, FAK and p-FAK so as to disrupt actin cytoskeleton and focal adhesions in HUVECs, indicating that endosulfan-induced endothelial injury in actin cytoskeleton structure was one of crucial mechanisms of increased endothelial permeability of HUVECs in transcellular pathway. The paracellular permeability of the endothelial barrier is maintained by the interendothelial junctions, including AJs and GJs. AJs are a type of cell-cell adhesion structure (Meng and Takeichi, 2009), connecting adjacent endothelial cells into the monolayer. Stress fibers play an active role in the maintenance of cadherindependent cell-cell contacts (Vasioukhin et al., 2000). Our data showed that endosulfan exposure reduced E-cadherin and b-catenin expression in HUVECs, indicating that endosulfan-induced disassembly of stress fibers might contribute to the effect of endosulfan on AJs. Besides the role in AJs, b-catenin acts as a transcriptional co-factor downstream of the Wnt signaling pathway, while cadherins have been proposed to sequester b-catenin at AJs and thereby modulate Wnt-mediated gene transcription (Balda and Matter, 2003). Our previous study found that endosulfan at only higher dose (60 mM) affected Wnt signaling in HUVECs (Xu

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Fig. 3. Involvement of LAMC1 in regulating endothelial function. (A) Representative Western blots; (B) Cell viability; (C) ROCK2 and PXN mRNA expression; (D) Endothelial permeability. The results are displayed as the mean ± SD (n ¼ 3) *P < 0.05, **P < 0.01 vs NC.

et al., 2016). We here observed no obvious changes in the expression levels of E-cadherin and b-catenin when HUVECs were exposed to endosulfan at 60 mM dose (Fig. 4A). We suppose that Wnt antagonists (MacDonald et al., 2009) or some upstream factors in the Wnt signaling pathway might influence the expression levels of E-cadherin and b-catenin. GJs are intercellular channels between apposed cell membranes and play a critical role in vascular biology including permeability, angiogenesis, and remodeling. Changes of GJs have been correlated to the development of atherosclerosis and hypertension (Brisset et al., 2009). GJs in vascular endothelial cells are mainly formed by the Cx37, Cx40 and Cx43 proteins (Morel et al., 2009). Evidence suggests that Cx43 acts as an atherogenic protein whereas Cx37 and Cx40 appear atheroprotective proteins (Pfenniger et al., 2013; Yuan et al., 2012). Previous studies demonstrated that Cx43 was downregulated in endothelial cells exposed to atherosclerosis and hypertension (Yeh et al., 2006). Regulation of Cx43-mediated GJIC is controlled by E-cadherin because cadherin-mediated cell-cell adhesion is a prerequisite for formation of GJs. Here, we found endosulfan inhibited gap junction activity in HUVECs via downregulation of Cx43, which might be associated with downregulation of E-cadherin-dependent cell-cell contacts. Reduced expression of Cx43 resulting in impaired GJIC is a potential indicator of endothelial dysfunction (Wang et al., 2008), thereby linked

to the increase in endothelial permeability induced by endosulfan. Evidence shows that excessive or inappropriate expression of MMPs may contribute to the pathogenesis of tissue destructive processes in a variety of diseases including human cancers, cardiovascular and cerebrovascular diseases (Nelson et al., 2000). Levels of MMP-1, MMP-2 and MMP-3 are all elevated in human atherosclerotic plaques (Newby, 2006). In our study, we found that endosulfan increased mRNA and protein expression of MMP-3, but MMP-1 protein appeared no change in HUVECs, indicating that MMP-3 might be involved in endosulfan-induced endothelial dysfunction through degrading its substrates. MMP-3 is regarded as a key genetic marker in pathological processes with broad substrate specificity, associated with various diseases (Munhoz et al., 2010). Laminin family is one of MMP-3 substrates, which assemble from a, b- and g-chain. They can control many cellular functions such as cell proliferation, adhesion, spreading and migration in both normal and pathologic processes (Tzu and Marinkovich, 2008). The laminin g-1 chain is the most ubiquitously expressed g subunit, encoded by LAMC1. Our results showed that LAMC1 had lower expression in endosulfan-exposed group than that of DMSO control group, indicating that endosulfan-induced MMP-3 overexpression might degrade laminin, resulting in the decrease in LAMC1 expression. It is reported that LAMC1 is a prerequisite for the brain

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Fig. 4. Effects of endosulfan on adherens junctions and gap junctions. (A) Expression of E-cadherin and b-catenin proteins. (B) Representative images of gap junction activity assay were shown, Scale bar, 25 mm. (C) Distance of scrape in different groups were quantified. (D) Cx43 mRNA expression. The results are displayed as the mean ± SD (n ¼ 3). *P < 0.05, **P < 0.01 vs DMSO.

Table 1 Prediction of human cardiovascular diseases in NextBio analysis of the 20 mM endosulfan dose group. Category

Score

Studies

Gene symbol

Accession No.

Fold change

1. Shock

71

9

2. Coronary arteriosclerosis (coronary atherothrombotic disease)

55

12

3. Disorder of cardiac function

55

17

46

24

NPL GPR84 MAP2K4 AHCTF1 NLRP3 SLC26A1 NPVF MON2 PPP1R12A PXN ROCK2 SPIN4 EDEM3 LPAR6 RAP2A ZNF518A ROCK2 SLC26A1 CCL5 ALOX12B SPRR1A LAMA4 PXN

NM_001200050 NM_020370 NM_003010 NM_015446 NM_001079821 NM_134425 NM_022150 BC111488 NM_002480 NM_001243756 NM_004850 NM_001012968 NM_025191 NM_016232 NM_021033 NM_014803 NM_004850 NM_134425 NM_002985 NM_001139 NM_005987 NM_001105207 NM_001243756

251.9 8.1 6.8 4.9 3.3 25.3 4.6 3.9 3.8 2.0 2.4 7.8 6.3 3.8 3.1 3.0 2.4 25.3 24.7 4.9 4.6 2.9 2.0

(hemorrhagic shock)

(coronary artery disease)

4. Hypertensive disorder (pulmonary arterial hypertension)

Note: Those genes in bold were emphasized to be associated with cytoskeleton in the present study. Common altered genes in at least two types of diseases were underlined.

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development, skin development and maturation after birth (Chen et al., 2009; Fleger-Weckmann et al., 2016). Knockdown of LAMC1 expression leads to early embryonic lethality (Mitchell et al., 2001), indicating that LAMC1 is important for bodies to keep stability and control growth. Our results also showed LAMC1 siRNAs reduced cell growth and enhanced endothelial permeability in HUVECs, suggesting that LAMC1 could interfere endothelial barrier function. To our knowledge, it is the first report that LAMC1 plays a critical role in regulating endothelial cell functions. Loss of contact with base membrane components such as laminin is likely to be responsible at least in part for the profound changes in gene expression (Newby, 2006). Laminins also influence intracellular pathway to regulate the cytoskeleton changes. It is reported that laminin-1 caused activation of FAK and mitogenactivated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways (Mruthyunjaya et al., 2010). We here found that knockdown of LAMC1 inhibited stress fiber formation and decreased the expressions of ROCK2 and PXN, as well as phosphorylation of FAK (Tyr397), similar with the results from endosulfan exposure. We suppose that LAMC1 might regulate FAK signaling pathway through activation of FAK, and endosulfan caused disruptions of actin cytoskeleton and focal adhesion through MMP3/LAMC1/FAK signaling pathway in HUVECs. There is epidemiological and experimental evidence linking POPs exposure to cardiovascular disease (Ha et al., 2007; La Merrill et al., 2013; Valera et al., 2013; Vena et al., 1998). Increased circulating levels of POPs have been associated with myocardial infarction (Ronn et al., 2011). Occupational exposure to dioxins and polychlorinated biphenyls (PCBs) has been correlated with an excess incidence of hypertension and heart diseases (Valera et al., 2013; Vena et al., 1998). Here, we predicted four cardiovascular diseases associated with endosulfan using those differentially expressed genes from microarray in Nextbio. The top one of these diseases is shock such as hemorrhagic shock. It is reported that increased vascular permeability was involved in hemorrhagic shock, leading to tissue edema, cell hypoxia and microcirculatory disturbance (Childs et al., 2010). Interestingly, we found that cytoskeleton related genes PXN and ROCK2 were predicted to be two of six key genes for coronary arteriosclerosis, while PXN and ROCK2 were key genes for pulmonary arterial hypertension and coronary artery disease, respectively. It is reported that ROCK2 was associated with the pathogenesis of atherosclerosis and cardiovascular disease (Mukai et al., 2001), while PXN was implied in pulmonary hypertension (Veith et al., 2012). These results strongly support the potential correlation between endosulfan and cardiovascular diseases.

5. Conclusions We here provide experimental evidence of the potential correlation between endosulfan and cardiovascular diseases through revealing molecular mechanism in increased endothelial permeability when exposure to endosulfan in HUVECs. We demonstrated that endosulfan enhanced endothelial permeability by transcellular and paracellular pathways, involving actin cytoskeleton and interendothelial junctions. Novel evidence suggested the involvement of LAMC1 in endosulfan-induced endothelial hyperpermeability. Our results from disease prediction illustrated that endosulfan might be relevant to human cardiovascular diseases. Importantly, a number of key genes such as cytoskeleton-related genes PXN and ROCK2 were screened out. Further investigations of the influence of those key genes will help develop specific treatments for endosulfaninduced cardiovascular diseases.

Acknowledgments The present work was supported by the National Natural Science Foundation of China (No. 21207012), “the Fundamental Research Funds for the Central Universities (3132014306, 3132016330)” and State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (No. KF2014-15).

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