2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin promotes endothelial cell apoptosis through activation of EP3/p38MAPK/Bcl‐2 pathway

Abstract Endothelial injury or dysfunction is an early event in the pathogenesis of atherosclerosis. Epidemiological and animal studies have shown that 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) exposure increases morbidity and mortality from chronic cardiovascular diseases, including atherosclerosis. However, whether or how TCDD exposure causes endothelial injury or dysfunction remains largely unknown. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to different doses of TCDD, and cell apoptosis was examined. We found that TCDD treatment increased caspase 3 activity and apoptosis in HUVECs in a dose‐dependent manner,at doses from 10 to 40 nM. TCDD increased cyclooxygenase enzymes (COX)‐2 expression and its downstream prostaglandin (PG) production (mainly PGE2 and 6‐keto‐PGF1α) in HUVECs. Interestingly, inhibition of COX‐2, but not COX‐1, markedly attenuated TCDD‐triggered apoptosis in HUVECs. Pharmacological inhibition or gene silencing of the PGE2 receptor subtype 3 (EP3) suppressed the augmented apoptosis in TCDD‐treated HUVECs. Activation of the EP3 receptor enhanced p38 MAPK phosphorylation and decreased Bcl‐2 expression following TCDD treatment. Both p38 MAPK suppression and Bcl‐2 overexpression attenuated the apoptosis in TCDD‐treated HUVECs. TCDD increased EP3‐dependent Rho activity and subsequently promoted p38MAPK/Bcl‐2 pathway‐mediated apoptosis in HUVECs. In addition, TCDD promoted apoptosis in vascular endothelium and delayed re‐endothelialization after femoral artery injury in wild‐type (WT) mice, but not in EP3−/− mice. In summary, TCDD promotes endothelial apoptosis through the COX‐2/PGE2/EP3/p38MAPK/Bcl‐2 pathway. Given the cardiovascular hazard of a COX‐2 inhibitor, our findings indicate that the EP3 receptor and its downstream pathways may be potential targets for prevention of TCDD‐associated cardiovascular diseases.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of the dioxins, whose toxicological effects are mediated by # These authors contributed equally to the work. *Correspondence to: Ying YU, M.D., Ph.D. E-mails: yuying@tmu.edu.cn or yuying@sibs.ac.cn interaction with the aryl hydrocarbon receptor (AhR), an orphan nuclear receptor [10]. Emerging epidemiological studies have shown that TCDD exposure increases the risk of cardiovascular diseases. For example, TCDD exposure leads to a higher incidence of hyperlipidaemia, as well as increased atherosclerotic plaques and intimamedia thickness [11,12]. In animal experiments, TCDD treatment has been shown to enhance atherosclerotic lesions in ApoE À/À mice [13,14]. However, the underlying mechanisms of TCDD-associated cardiovascular diseases are not fully understood. Previous studies have reported that TCDD causes apoptosis in many types of cells, including chondrocytes [15], lymphoblastic T cells [16] and pheochromocytoma cells [17]. Whether TCDD exposure promotes endothelial cell apoptosis has not been determined.
Prostaglandins (PGs) are implicated in the regulation of endothelial homeostasis [18]. Cyclooxygenase enzymes (COX-1 and COX-2) are the rate-limiting enzymes for the conversion of arachidonic acid (AA) to downstream PGs. COX-2 inhibitors have been shown to improve vascular function in diabetic rats by suppressing vascular endothelial growth factors [19], meanwhile high glucose promotes human umbilical vein endothelial cells (HUVECs) apoptosis by up-regulating of COX-2 through activation of NF-кB signalling [20]. Interestingly, TCDD exposure increases COX-2 mRNA levels in HUVECs [6], mouse lung endothelial cells (MLECs) [6], rat hepatocytes [21] and mouse fibroblasts [22]. We envision that TCDD exposure may elicit EC apoptosis by inducing COX-2 expression and its downstream PG products. Accumulated evidence has shown that COX-2-derived PGs and downstream PG receptors play important roles in cellular apoptosis. For example, increased mast cell apoptosis by PGE 2 is dependent on an increase in E-prostanoid receptor (EP) subtype 3 (EP3) activation, intracellular calcium release and calmodulin-dependent kinase II and MAPK activation [23]. The role of PG and its receptors in TCDD-caused endothelial cell apoptosis has yet to be determined.
This study aimed to determine whether TCDD causes apoptosis in endothelial cells and to explore the potential mechanisms involved. Our data showed that HUVEC apoptosis caused by TCDD depends on COX-2 activity. Inhibition of COX-2 and the PGE 2 receptor subtype EP3 suppressed apoptosis in TCDD-treated HUVECs. Moreover, activation of the EP3 receptor increased Rho activity and p38 MAPK phosphorylation and decreased Bcl-2 expression in TCDD-treated HUVECs, while suppression of Rho and p38 MAPK activity abrogated increased apoptosis in HUVECs stimulated by TCDD. Additionally, TCDD exposure reduced EP3 receptor-dependent re-endothelialization of femoral arteries after targeted injury in mice.

Animals
EP3 knockout (EP3 À/À ) mice and wild-type (WT) C57BL/6 were used [24]. Femoral arteries of 10-to 12-week-old male mice were injured using a wire 0.38 mm in diameter (Cook Group Inc., Bloomington, IN, USA). Then, the mice were injected with 10 lg TCDD/kg bodyweight intraperitoneally every other day. On the tenth day, the injured femoral arteries were collected for further examination. All animal procedures were approved by the institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Chinese Academy of Sciences.

Cell viability assay
HUVECs were seeded into 96-well plates at the same cell number per well (5 9 10 3 /well). Once the cells reached 50% confluence, they were pre-treated with various inhibitors for 24 hrs and incubated with TCDD at indicated concentrations for an additional 24 hrs. At the end of each culture, the medium was removed and replaced with 100 ll fresh medium and 10 ll Cell Counting Kit-8 (CCK-8) Assay Kit reagent (Dojindo, Kumamoto, Japan). Plates were incubated at 37°C for 2 hrs. Next, absorbance was measured at 450 nm using a Spec-traMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The reference wavelength was 600 nm. Background absorbance (from wells without cells) was subtracted from all values, and data are presented as relative values to controls as previously described [25]. All experiments were performed in triplicate with 3-5 repeats per culture.

Cell apoptosis detection
Apoptosis was assessed with an Annexin V-FITC/Propidium Iodide (PI) apoptosis detection kit (Dojindo) using a BD FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, HUVECs were seeded into 6-well plates and incubated with various inhibitors for 24 hrs and then stimulated with TCDD for an additional 24 hrs. The cells were then harvested and washed once in phosphate-buffered saline (PBS) and once in 19 binding buffer. After resuspension in 19 binding  buffer, we added 5 ll Annexin V to 100 ll cell suspension. Next, the mixture was incubated for 15 min. at room temperature, washed and resuspended in 19 binding buffer. After addition of 5 ll PI dye, HUVECs were analysed by flow cytometry. In addition, caspase 3 activity was assayed using a Caspase 3 Activity Assay Kit (Beyotime, Haimen, China) according to the manufacturer's instructions. Data are shown as relative values to controls and representative of at least three independent experiments [20].

DAPI staining
The procedure was performed as described previously [26]. In brief, HUVECs were washed twice with PBS. Next, the cells were fixed with 3.7% paraformaldehyde for 10 min. at room temperature and washed twice with PBS. Afterwards, the cells were stained with DAPI (0.5 g/ ml) for 1 hr in the dark. The stained cells were washed with PBS and examined by fluorescence microscopy. Apoptotic cells were identified by condensation and fragments of nuclei.

RNA interference and Bcl-2 overexpression
Cells were transiently transfected with 100 pmol AhR nuclear translocator (ARNT), EP1-4 receptor or scrambled siRNA per well in 6-well plates, or 5 pmol EP1-4 receptor or scrambled siRNA per well in 96well plates using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Three pairs of siRNA specific sequences for ARNT and EP1-4 mRNA were designed by Shanghai GenePharma Co., Ltd. (Shanghai, China). The most effective sequences are shown in Table S1. In addition, cells were seeded into 6-well plates or 96-well plates, and transfected with Bcl-2-pcDNA 3.1 plasmid (Addgene) or vector using Lipofectamine 2000 reagent (Invitrogen). All analyses were performed for 24 hrs after transfection and TCDD stimulation.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNAs were isolated from HUVECs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and treated with DNase I (Promega, Madison,WI, USA). RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). RNA (2.0 lg) was reverse-transcribed with Reverse Transcription Reagent kits and oligo (dT) 18 primers (Takara Bio, Inc. Dalian, China) as recommended. qRT-PCR was performed using a CFX96 TM real-time PCR detection system (Bio-Rad, Hercules, California, USA) and iQ TM SYBR Green Supermix (Bio-Rad) as described by the manufacturer. Gene expression was normalized to the internal control, GAPDH and presented as relative levels calculated by the 2 DD Ct method [27]. All specific primers for the genes investigated are listed in Table S1.

Rho GTPase activity assay
HUVECs were grown to confluence on 15 cm 2 dishes, and equal numbers of serum-starved cells were incubated with TCDD (40 nM) or DMSO in RPMI 1640 for 3 min. at 37°C. The cells were washed with cold PBS and then harvested in ice-cold lysis buffer. RhoA activation assay kits were purchased from Cell Biolabs (San Diego, CA, USA) and used to detect active RhoA according to the manufacturer's protocol.

PG extraction and analysis
HUVECs were treated with DMSO, 20 lM NS-398, 40 nM TCDD or a combination of TCDD with 20 lM NS-398 for 24 hrs, after which the medium was changed to the medium containing fresh FBS and 30 lM arachidonic acid (AA) for 15 min. The medium was collected for PG production analysis with liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS; Agilent Technologies, Santa Clara, California, USA) and normalized to total protein. The detailed procedure was routinely performed in our laboratory [28].

Femoral artery wire injury model
The femoral artery wire injury model has been described previously elsewhere [28]. In brief, mice were anesthetized with isoflurane during surgery. The left femoral arteries were exposed by blunt dissection and monitored under a surgical microscope. After the distal artery was encircled with a 5-0 silk suture, a vascular clamp was placed near the inguinal ligament, and a guide wire (0.38 mm in diameter; Cook Group Inc., Bloomington, IN, USA) was inserted into the arterial lumen through an arteriotomy made in the distal perforating branch. The guide wire was left in place for 3 min. to denude the artery. Next, the wire was removed, and the silk suture was released to restore blood flow. The skin incision was closed with a 6-0 silk suture. Mice were allowed to recover, and femoral arteries were harvested 10 days post-injury.

TUNEL and Evans blue staining
To examine whether TCDD is involved in apoptosis and re-endothelialization in vivo, we employed TUNEL staining and the Evans blue dye methods [29,30]. Briefly, the mice were injected intravenously with 0.5 ml of 0.5% Evans blue dye (Sigma-Aldrich) in PBS on the tenth day after vascular injury. The injured femoral arteries were then incised longitudinally to expose the luminal surface and fixed in 4% polyformaldehyde solution for 5 min. Planimetric analysis was performed to calculate the ratio of the re-endothelialized area, defined as an area not stained with Evans blue relative to the entire luminal surface area of injured arteries. In addition, frozen sections of the femoral arteries were prepared; then, DNA fragments were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay with an Apoptosis Detection Kit (Yesen, Shanghai, China) according to the manufacturer's protocols.

Statistical analysis
Data are expressed as means AE S.E.M. P values were calculated by a two-tailed Student's t-test or one-or two-way ANOVA with Bonferroni's post hoc analyses as appropriate (GraphPad Prism 5.0 software). P < 0.05 was considered statistically significant.

TCDD promotes apoptosis in HUVECs
We first examined the apoptosis, cell viability, caspase 3 activity and apoptotic morphology of HUVECs in response to TCDD stimulation. As shown in Figure 1A and B, PI-an indicator of late apoptosis-and Annexin V-FITC co-staining was performed to examine the apoptosis. We observed that TCDD caused apoptosis in a dose-dependent manner (P < 0.05), and the apoptosis of the group treated with 40 nM TCDD (26.24 AE 2.19%) was nearly threefold (P < 0.05) higher than that of the control group (9.67 AE 1.55%). We also found that 40 nM TCDD significantly decreased (P < 0.05) the cell viability from 100% to 76.98% (Fig. 1C). As caspase 3 activation is an indicator of cell apoptosis, we examined the caspase 3 activity in HUVECs treated by TCDD at different concentrations and exposure times, and observed more than twofold increases of caspase 3 activity (P < 0.05; Fig. 1D and E) at 40 nM TCDD after 24 hrs. Meanwhile, TCDD promoted condensation and fragmentation of nuclei in HUVECs as seen by DAPI staining (P < 0.05; Fig. 1F and G), further confirming that TCDD leads to apoptosis in HUVECs.

COX-2 is involved in TCDD-caused apoptosis of HUVECs
COX-1 and COX-2 are the key enzymes in PG biosynthesis. COX-2 protein expression was increased in HUVECs exposed to TCDD, whereas COX-1 protein levels were not markedly altered ( Fig. 2A). TCDD also increased mRNA levels of CYP1A1 and COX-2, which peaked 4-8 hrs after TCDD challenge (P < 0.05; Fig. S1A and B).
TCDD can stimulate COX-2 expression through both a nongenomic pathway [31] or activation of the AhR/ARNT heterodimeric transcription factor [32]. We further investigated whether TCDDcaused COX-2 expression in ECs occurs through direct activation of the AhR/ARNT transcription complex. We observed that increases in COX-2 mRNA and protein expression after TCDD treatment were abolished in HUVECs by knockdown of the ARNT gene ( Fig. S1D and E), indicating that TCDD caused COX-2 expression in ECs through binding to the AhR and activation of the AhR/ARNT transcription complex. In addition, TCDD significantly reduced HUVEC viability (P < 0.05, Fig. 2B), this decline was abrogated by a selective COX-2 inhibitor, NS-398 (P < 0.05), but not by the COX-1 inhibitor SC-560 (Fig. 2B). Moreover, NS-398 also reduced the increased apoptosis (P < 0.05; Fig. 2C and D) and caspase 3 activity (P < 0.05; Fig. 2E) caused by TCDD treatment. Thus, these results suggest that COX-2 is involved in TCDD-caused HUVEC apoptosis.

COX-2-derived PGE 2 facilitates TCDD-caused apoptosis in HUVECs via the EP3 receptor
We next assessed PG production in TCDD-treated HUVECs and found that TCDD-caused increases of COX-2 expression in HUVECs were associated with a higher production of PGE 2 and PGI 2 (measured as its stable hydrolyzed product, 6-keto-PGF 1a ) in culture, which could be blocked by NS-398 (P < 0.05; Fig. 3A and B). However, production of other PGs (PGD 2 , PGF 2a and TxB 2 ) was not markedly altered by treatment with TCDD or the COX-2 inhibitor NS-398 ( Fig. S2A-C). Expression of PGI 2 receptor-the IP was unchanged in HUVECs after TCDD exposure (Fig. S3A), and IP antagonist CAY10441 had no   significant effects on the increased caspase 3 activity and reduced viability of TCDD-treated HUVECs ( Fig. S3B and C). To further identify which PGE 2 receptor mediates HUVEC apoptosis caused by TCDD, both pharmacological inhibitors and specific siRNAs targeting EP1-4 receptors were used to pre-treat HUVECs followed by TCDD stimulation. As shown in Figure 4, TCDD-caused apoptosis in HUVECs, evidenced by cell viability and caspase 3 activity measurements, was significantly attenuated by the EP3 inhibitor, L798106 (P < 0.05; Fig. 4A-C) and EP3 siRNA (P < 0.05, Fig. 4D-F), suggesting that the EP3 receptor plays a pivotal role in TCDD-mediated apoptosis in ECs. In addition, we determined the transcript levels of EP1-4 in HUVECs exposed to TCDD and found that only EP3 mRNA expression was elevated notably after 40 nM TCDD challenge (P < 0.05; Fig. S4A-D).
The EP3-dependent p38MAPK/Bcl-2 pathway is activated in TCDD-treated HUVECs MAPK is involved in regulating the proliferation, survival and cell death responses of many cell types [33,34]. To explore whether intracellular JNK, p38 MAPK and ERK signalling pathways were involved in enhanced cell apoptosis in response to TCDD exposure, a series of experiments were performed using the MAPK subtypes inhibitors. Among them, the p38 MAPK inhibitor, SB203580, abrogated TCDD caused HUVEC apoptosis (P < 0.05; Fig. 5A and B). HUVEC viability was reduced~30% by TCDD exposure, which was restored by treatment with SB203580 (P < 0.05; Fig. 5A). Meanwhile, TCDD stimulation elevated caspase 3 activity in HUVECs to approximately 2.5-fold of the control (P < 0.05), which was diminished by SB203580 treatment (P < 0.05; Fig. 5B). The similar results were observed for another p38 MAPK inhibitor SB202190 (Fig. 5C), indicating that TCDD treatment results in HUVEC apoptosis via p38 MAPK signalling. Apoptosis is regulated precisely by the interaction between pro and anti-apoptotic Bcl-2 family proteins. Primary anti-apoptotic proteins include Bcl-2, Bcl-XL and Mcl-1, while pro-apoptotic proteins are composed of Bax, Bak, Bim, Bid, Bad and Bik [35]. We therefore measured the mRNA levels of Bad, Bak1, Bax, Bcl-2 and Bim and found that only Bcl-2 transcript levels decreased in TCDD-treated HUVECs (P < 0.05; Figure 6A and Fig. S5A-D). Similarly, Bcl-2 protein expression also was greatly inhibited by TCDD treatment as compared to the vehicle (Fig. 6B). In addition, reduction in cell viability and elevation of caspase 3 activity by TCDD treatment were reversed completely in HUVECs by exogenous Bcl-2 overexpression (P < 0.05; Fig. 6C-E). Then, we examined whether Bcl-2 is the downstream target gene of the EP3/ p38MAPK axis, which regulates TCDD-caused apoptosis. We found that TCDD promoted p38 MAPK phosphorylation and decreased Bcl-2 protein expression, while the p38 inhibitor SB203580 recovered Bcl-2 expression levels (Fig. 6F). Furthermore, EP3 inhibition by L798106 suppressed the augmented p38 MAPK phosphorylation and subsequently restored Bcl-2 protein expression in TCDD-treated HUVECs (Fig. 6G), further supporting activation of EP3/p38MAPK/ Bcl-2-mediated TCDD-triggered apoptosis in HUVECs.
Activated RhoA is required for EP3-mediated apoptosis of HUVECs caused by TCDD As the EP3 receptor is linked to the small GTP-binding protein Rho [24,28], and Rho activates the p38 MAPK pathway [36][37][38], we investigated whether Rho mediates p38 activation in TCDD-elicited apoptosis in HUVECs. Non-radioactive Rho pull-down assay showed that RhoA activity was minimal in unstimulated HUVECs, but was greatly increased following TCDD treatment (Fig. 7A). Interestingly, Rhoassociated protein kinase (ROCK) blocker Y26732 attenuated the reduction in cell viability and activation of caspase 3 in HUVECs following TCDD challenge (P < 0.05; Fig. 7B and C). In addition, Y27632 restored the altered p38 MAPK phosphorylation levels and Bcl-2 protein levels in TCDD-treated HUVECs (Fig. 7D), implying that RhoA/p38MAPK/Bcl-2 signalling participates in apoptosis in HUVECs caused by TCDD. Furthermore, EP3 receptor knockdown suppressed activation of RhoA activity and p38MAPK phosphorylation and restored the reduced Bcl-2 protein levels in TCDD-treated HUVECs (Fig. 7E). Taken together, we conclude that TCDD promotes HUVEC apoptosis through the COX-2/PGE 2 /EP3/RhoA/p38MAPK/Bcl-2 pathway (Fig. 7F). TCDD exposure suppresses re-endothelialization of wire-injured femoral arteries by activating the EP3 receptor in mice To further test whether the EP3 receptor participates in apoptosis and re-endothelialization in vivo, WT and the EP3 receptor knockout mice were injured in the femoral arteries and injected intraperitoneally with TCDD. Then, the apoptosis and re-endothelialization in injured vessels were examined using a TUNEL kit and Evans blue staining, respectively. Ten days after the injury, TCDD injection resulted in more apoptosis in the vascular endothelium of WT mice than that of EP3 À/ À mice (Fig. 8A). In agree with apoptosis, the degree of re-endothelialization in WT mice, but not in EP3 À/À mice, was significantly lower after TCDD injection than after the vehicle injection (P < 0.05; Fig. 8B and C).

Discussion
TCDD elicits a broad spectrum of toxic and biochemical responses including a wasting syndrome, tumour promotion, teratogenesis, hepatotoxicity, modulation of endocrine systems, immunotoxicity and cardiovascular diseases [39,40]. Many of these effects are mediated via the AhR activation [41]. Epidemiological and animal studies have shown that TCDD exposure contributes to pathogenesis of cardiovascular diseases [11,13,42]. In this study, we found that TCDD promoted endothelial apoptosis through the COX-2/PGE 2 /EP3/p38MAPK/ Bcl-2 pathway and impaired vascular re-endothelialization after injury in mice through the EP3 receptor.
COX catalyses the first step in the formation of PGs from AA. Two isoforms of COX are often referred to as COX-1 and COX-2. Notably, COX-2 plays an important role in the regulation of cell survival and proliferation. In human osteosarcoma cells, overexpression of the COX-2 gene has been shown to suppress proliferation and promote apoptosis [43]. High glucose levels induce COX-2 expression and facilitate apoptosis in HUVECs [20]. Similarly, we observed that TCDD treatment increased HUVEC apoptosis in a dose-dependent manner by up-regulating COX-2 expression. However, COX-2 promotes cell proliferation and survival in some cancer cell lines [44][45][46]. These discrepancies may be ascribed to different functions of multiple PGs and receptors downstream of COXs [47,48]. There are four subtypes of the PGE 2 receptor (EP1-4). We found that TCDD caused HUVEC apoptosis by activating the COX-2/PGE 2 /EP3 axis. Similarly, the EP3 receptor also mediates apoptosis in neurons in the murine ischemic cortex [49].
The MAPK family contains JNK, p38 MAPK and ERK. Extensive studies demonstrated a critical role for MAPK signalling pathways in the regulation of various cellular processes, including migration, proliferation, differentiation, development, apoptosis and cell cycle arrest [50]. We assessed whether intracellular JNK, p38 MAPK and ERK signal pathways were involved in enhanced cell apoptosis in response to TCDD exposure and revealed that only the p38 MAPK inhibitor, SB203580, attenuated TCDD-caused apoptosis in HUVECs. The increased p38 MAPK phosphorylation by TCDD exposure was blocked by SB203580. Several studies have shown that TCDD-caused apoptosis depends on activation of different MAPK pathways in various cell types. In the RAW 264.7 cells, both p38 MAPK and ERK pathways mediate TCDD-caused apoptosis [51]. However, JNK and ERK signalling are activated in TCDD-caused Jurkat T lymphocyte cell apoptosis [52]. Li et al., [34] demonstrated that TCDD could lead to apoptosis in rat primary cortical neurons through mediating p38 MAPK and JNK pathways.
The Bcl-2 protein family regulates apoptosis and contains both anti-apoptotic (Bcl-2, Bcl XL, and Mcl 1) and pro-apoptotic proteins (Bax, Bak, Bim, Bid, Bad and Bik) [35]. We found that Bcl-2 mRNA and protein expression were markedly reduced in TCDD-treated HUVECs. Forced expression of the Bcl-2 gene attenuated TCDD-caused apoptosis of HUVECs, along with suppression of caspase 3 activity. In agreement with our observations, TCDD also promotes apoptosis of human trophoblast-like JAR cells [53] and bovine MDBK cells [54] by down-regulating expression of the Bcl-2 gene. Given that EP3 inhibition or knockdown restored TCDD-caused-p38 MAPK activation and suppressed Bcl-2 expression, EP3-mediated activation of the p38MAPK/Bcl-2 pathway contributes to apoptosis increased by TCDD in HUVECs. Rho activates p38 MAPK [36][37][38] and the EP3 receptor couples small G protein and activates RhoA [24,28]. Indeed, we demonstrated that TCDD exposure increased p38 MAPK phosphorylation by activating EP3/Rho signalling in HUVECs.
In summary, TCDD exposure increases endothelial apoptosis through the COX-2/PGE 2 /EP3/p38MAPK/Bcl-2 pathway and delays arterial re-endothelialization after injury through activation of the EP3 receptor. These findings suggest that blocking the EP3 receptor may confer cardiovascular protection against TCDD toxicity.