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Deep vein thrombosis (DVT) is a common complication in hematologic malignancies and immunologic disorders. Endothelial cell injury and dysfunction comprise the critical contributor for the development of DVT. A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), a plasma metalloprotease that cleaves von Willebrand factor, acts as a critical regulator in normal hemostasis. This study was aimed to explore the role of ADAMTS13 in endothelial cell injury during DVT and the possible mechanism. First, human umbilical vein endothelial cells (HUVECs) were exposed to hydrogen peroxide (H2O2). Then, the mRNA and protein expressions of ADAMTS13 were evaluated with the reverse transcription-quantitative polymerase chain reaction and western blot. After treatment with recombinant ADAMTS13 (rADAMTS13; rA13), the viability and apoptosis of H2O2-induced HUVECs were assessed by cell counting kit-8 assay and terminal-deoxynucleoitidyl transferase-mediated nick end labeling staining. In addition, the levels of prostaglandin F1-alpha, endothelin-1, and reactive oxygen species were detected using the enzyme-linked immunosorbent assay and dichloro-dihydro-fluorescein diacetate assay. The expressions of proteins related to p38/extracellular signal-regulated kinase (ERK) signaling pathway were estimated with the western blot. Then, p79350 (p38 agonist) was used to pretreat cells to analyze the regulatory effects of rA13 on p38/ERK signaling in H2O2-induced HUVEC injury. The results revealed that ADAMTS13 expression was significantly downregulated in H2O2-induced HUVECs. The reduced viability and increased apoptosis of HUVECs induced by H2O2 were revived by ADAMTS13. ADAMTS13 also suppressed the oxidative stress in HUVECs after H2O2 treatment. Besides, ADAMTS13 was found to block p38/ERK signaling pathway, and p79350 reversed the impacts of ADAMTS13 on the damage of HUVECs induced by H2O2. To sum up, ADAMTS13 could alleviate H2O2-induced HUVEC injury through the inhibition of p38/ERK signaling pathway.
Keywords: ADAMTS13, apoptosis, deep vein thrombosis, oxidative stress, p38/ERK signaling pathway
How to cite this URL:
Zheng G, Zhang Q, Li C, Fan W, Pan Z, Zhou Y, Chen Y, Rong J. ADAMTS13 inhibits H2O2-induced human venous endothelial cell injury to attenuate deep-vein thrombosis by blocking the p38/ERK signaling pathway. Chin J Physiol [Epub ahead of print] [cited 2023 Nov 30]. Available from: https://www.cjphysiology.org/preprintarticle.asp?id=389335 |
Guangfeng Zheng and Qiang Zhang contributed equally to this work.
| Introduction | |  |
As a life-threatening disease, deep vein thrombosis (DVT), which is associated with the formation of blood clots in the deep venous system, is especially prevalent in the pediatric population.[1],[2] It is reported that DVT can cause devastating consequences such as pulmonary embolism, postthrombotic syndrome, and death.[3] There are many risk factors contributing to the occurrence of DVT, including obesity, prolonged hospitalization, major surgery, mechanical compression as well as systemic conditions, such as malignancy.[4],[5] As is known to all, the symptoms of DVT range from none to pain and swelling in the legs and currently the major therapy for DVT is the application of blood-thinning drugs (anticoagulation) and mechanical intervention.[5] In recent years, the potential of targeted treatments for DVT has been highlighted.[6] Nevertheless, the hidden reaction mechanism of DVT still needs further elucidation.
A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), which is a member of ADAMTS family with characteristic ADAM-like protease domains, is a critical protease in the regulation of ultra-large von Willebrand factor.[7] It is reported that ADAMTS13 is involved in many diseases, including ischemic stroke, myocardial infarction, along with kidney disease.[8] In addition, it also plays pivotal roles in regulating obesity, inhibiting inflammatory response, degrading matrix, remodeling tissue, and promoting angiogenesis.[9],[10],[11] Yuko and coworkers claimed that ADAMTS13 depletion led to increased venous thrombosis in mice.[12] Interestingly, ADAMTS13 activity was testified to be low in patients suffering from DVT and lower ADAMTS13 activity increased the risk of DVT.[13]
Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), which are two critical subgroups of MAPK, are the vital mediators in regulating cell processes, such as cell proliferation as well as apoptosis.[14],[15] Besides, a case of study has well documented that rhADAMTS13 could downregulate the phosphorylation of p38/ERK.[16] Interestingly, the suppression of p-p38, p-JNK, and p-ERK decreased the expressions of thrombosis-related markers in H2O2-induced HUVECs.[17] Nevertheless, the relationship between ADAMTS13 and p38/ERK in DVT has not been clarified so far.
Endothelial cell injury and dysfunction are the major factors contributed to DVT. Previous studies reported that excessive oxidative stress is a common cause of vascular endothelial cell injury.[18],[19] Moreover, intracellular reactive oxygen species (ROS) can aggravate apoptosis in vascular endothelial cells and decrease antiapoptotic molecule expression.[20] Hydrogen peroxide (H2O2) is widely applied to induce HUVECs to establish DVT model.[17],[21] This paper was intended to reveal the functions of ADAMTS13 on the proliferation, apoptosis, and oxidative stress in H2O2-induced HUVECs and discuss the hidden reaction mechanism.
| Materials and Methods | | |
Cell culture and treatment
Human umbilical vein endothelial cells (HUVECs) that provided by BeNa Culture Collection (Henan, China) were incubated in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) which was decorated with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences) and 1% penicillin-streptomycin (Beyotime, Shanghai, China) at 37°C with 5% CO2. To establish oxidative stress injury model in vitro, HUVECs were exposed to 0.5 mM H2O2 (Sigma-Aldrich, Merck KGaA) for 24 h.[21] To evaluate the effect of ADAMTS13 on HUVEC injury exposed to H2O2, the HUVECs were pretreated with 1, 4, or 8 nM recombinant human ADAMTS13 (rADAMTS13; rA13; R&D systems, Minneapolis, MN, USA) for 30 min prior to H2O2 treatment.[22],[23] To figure out the mechanism of p38/ERK signaling pathway, p79350 (p38 agonist; Invitrogen, Carlsbad, CA, USA) was used to pre-treat HUVECs for 1 h and then treated with or without rA13 at concentrations of 1, 4, or 8 nM for 30 min before H2O2 treatment.[24]
Western blot
Total proteins were extracted from HUVECs with RIPA lysis buffer (Solarbio), following which was the quantification with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc.). Separated with 8% sodium dodecyl sulfate/polyacrylamide gel electrophoresis, equal amounts of proteins (20 μg per lane) were transferred onto PVDF membranes (MilliporeSigma). Then, the membranes were impeded by 5% nonfat milk. The membranes were subsequently incubated with primary antibodies specific to ADAMTS13 (ab177940; 1:1000; Abcam), NADPH oxidases 2 (Nox2; ab129068; 1:5000; Abcam), NADPH oxidase 4 (Nox4; ab154244; 1:1000; Abcam), phosphor (p)-p38 (ab178867; 1:1000; Abcam), p-ERK 1/2 (ab201015; 1:1000; Abcam), p38 (ab170099; 1:1000; Abcam), ERK1/2 (ab184699; 1:10000; Abcam) or GAPDH (ab9485; 1:2500; Abcam) overnight at 4°C, after which was the incubation with HRP-labeled secondary antibody (ab6721; 1:2000; Abcam) at the room temperature for 2 h. Finally, the protein bands were visualized with ECL Detection Reagent (Yeasen Biotech) and the density was analyzed with ImageJ software (Version 146).
Reverse transcription-quantitative polymerase chain reaction
Total RNA was extracted from sample HUVECs using TRIzol reagent (Thermo Fisher Scientific, Inc.) and then reverse transcribed into complementary DNA (cDNA) with a commercial RevertAid™ cDNA Synthesis kit (Bio-Rad) in light of the recommended specifications. The cDNA templates were amplified by SYBR Green polymerase chain reaction (PCR) Master Mix (Takara, Toyobo, Japan) on the 7500 Fast Real-time PCR (RT-PCR) system strictly according to the manufacturer's instructions. The sizes of the PCR products were 150 bp. The relative gene expression of ADAMTS13 was calculated with comparative Ct method.[25] The following were the primer sequences: ADAMTS13 forward primer: 5'-TCCTTGCCTGTGGCTTTCTC-3', reverse primer: 5'-TCCTTGCCTGTGGCTTTCTC-3' or GAPDH forward primer: 5'-AATGGGCAGCCGTTAGGAAA-3', reverse primer: 5'-GCGCCCAATACGACCAAATC-3'.
Cell counting kit-8 assay
The viability of HUVECs was assessed with CCK-8 assay. Initially, HUVECs were injected into 96-well plates at a density of 5 × 103 cells/well and then incubated for 24 h. After the indicated treatment, 10 μL CCK-8 reagent (Beyotime, Shanghai, China) was added into each well to further incubate cells for another 2 h. The optical density at 450 nm was detected by means of a microplate reader (Thermo Fisher Scientific, Inc.).
Terminal-deoxynucleotidyl transferase-mediated nick end labeling
The apoptosis of HUVECs was estimated with a TUNEL assay kit (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, after the indicated treatment, HUVECs were subjected to immobilization with 4% paraformaldehyde at room temperature for 15 min and permeation with 0.25% Triton-X100 at room temperature for 20 min. Subsequently, HUVECs were rinsed by PBS and then incubated with TUNEL reaction solution for 1 h according to the manufacturer's instructions. Afterward, DAPI was employed for the counterstaining of cell nuclei in the dark. The counting of apoptotic cells in five randomly selected fields was implemented under a florescent microscope (Olympus Corporation).
Enzyme-linked immunosorbent assay
To determine the release of thrombogenic factors, ELISA kits (R&D Systems, MN, USA) were performed to estimate the levels of prostaglandin F1-alpha (PGF1α) and endothelin-1 (ET-1) according to the manufacturer's instructions. The optical density was detected at 540 nm with a microplate reader (Thermo Fisher Scientific, Inc.).
Measurement of reactive oxygen species
With the application of ROS Assay Kit (Nanjing Jiancheng Bioengineering institute, Nanjing, China), the content of ROS in cell suspension was appraised in light of standard protocol. Brief, after the indicated treatment, HUVECs were counterstained with 5 μmol/l dichloro-dihydro-fluorescein diacetate at 37°C for 30 min in the dark, after which were the rinse and resuspension. Finally, the relative level of ROS was evaluated.
Statistical analysis
All experiments were independently conducted in triplicate. The collected data were presented in the format of mean ± standard deviation and analyzed with GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA). The differences between two groups were compared with Student's t-test while one-way analysis of variance with Tukey's post hoc test was employed for the demonstration of comparisons among multiple groups. It was supposed to be statistically significant when P < 0.05.
| Results | | |
ADAMTS13 expression was decreased in H2O2-treated HUVECs
H2O2 is widely applied to induce HUVECs to simulate endothelial cell injury during DVT.[17],[21] First, HUVECs were treated with H2O2 for 24 h, and ADAMTS13 expression was evaluated by the reverse transcription-quantitative PCR (RT-qPCR) and western blot. As presented in [Figure 1]a and [Figure 1]b, the mRNA and protein expressions of ADAMTS13 in HUVECs were conspicuously reduced after H2O2 treatment when compared with that in control group. These results suggested that the abnormal ADAMTS13 expression might be related to DVT. | Figure 1: A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) expression was decreased in hydrogen peroxide (H2O2)-treated human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to 0.5 mM H2O2 for 24 h. (a) The mRNA expression of ADAMTS13 was detected using reverse transcription-quantitative polymerase chain reaction. (b) The protein expression of ADAMTS13 was detected using western blot. Data from three independent replicates were presented as mean ± standard deviation. **P < 0.01 and ***P < 0.001.
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ADAMTS13 inhibited apoptosis and thrombosis-related factor expression in H2O2-treated HUVECs
Then, to investigate the effects of ADAMTS13 on HUVEC injury after H2O2 exposure, the rA13 with concentrations of 1, 4, or 8 nM was applied for the pretreatment of HUVECs for 30 min before H2O2 exposure.[22] Cell viability was assessed by CCK-8 assy. Relative to the control group, the viability of HUVECs was greatly reduced by H2O2 treatment. Compared with the H2O2 group, the decreased viability in H2O2-treated HUVECs was partially revived by rA13 treatment [Figure 2]a. Results obtained from TUNEL assay kit revealed that the promoted apoptosis in HUVECs because of H2O2 induction was markedly reduced following the treatment of rA13 [Figure 2]b and [Figure 2]c. Then, the effect of rA13 treatment on thrombosis-related factor expression in HUVECs exposed to H2O2 was detected. As [Figure 2]d and [Figure 2]e depicted, H2O2 stimulation markedly reduced the expression of vasodilatation-related factor PGF1α, a stable metabolite of PGI2, and elevated the expression of transcripts of ET-1, a critical regulator for vasoconstriction. Importantly, ADAMTS13 reversed the effects of H2O2 on the expression of PGF1α and ET-1. The above results revealed that ADAMTS13 restrained apoptosis and thrombosis-related factor expression in H2O2-treated HUVECs. | Figure 2: A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) inhibited hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to 0.5 mM H2O2 for 24 h with or without 1, 4 or 8 nM rA13 pretreatment for 30 min. (a) The viability of HUVECs was detected using CCK-8 assay. (b) The apoptosis of HUVECs was detected using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). (c) Quantification of apoptotic cells in TUNEL staining. (d) The content of PGF1α was detected using enzyme-linked immunosorbent assay (ELISA). (e) The content of ET-1 was detected using ELISA. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001.
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ADAMTS13 inhibited oxidative stress in H2O2-induced HUVECs
Endothelial cell injury under oxidative stress is the key regulator in the development of DVT by evoking prothrombosis response.[26] The level of oxidative stress was assessed in the subsequent experiments. Relative to the control group, H2O2 treatment remarkably elevated the level of ROS, while rA13 treatment exhibited the opposite effects on it, evidenced by the reduced level of ROS in H2O2-treated HUVECs with rA13 treatment [Figure 3]a. As [Figure 3]b depicted, the elevated protein expressions of Nox2 and Nox4 in HUVECs due to H2O2 induction were conspicuously reduced following the treatment of rA13, suggesting the suppressive effects of rA13 on oxidative stress in H2O2-induced HUVECs [Figure 3]b. | Figure 3: A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) inhibited oxidative stress in hydrogen peroxide (H2O2)-induced human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to 0.5 mM H2O2 for 24 h with or without 1, 4 or 8 nM rA13 pretreatment for 30 min. (a) The level of reactive oxygen species (ROS) was detected using ROS assay kit. (b) The expressions of Nox2 and Nox4 were detected using western blot. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001.
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ADAMTS13 blocked p38/ERK signaling pathway
A sustained increase in ROS can promote endothelial cell apoptosis through the activation of the MAPK signaling pathway.[27] The expressions of p38/ERK signaling pathway-related proteins were assessed with western blot and the results demonstrated that H2O2 induction greatly elevated the expressions of p-p38 and p-ERK1/2 in comparison with those in the control group, while rA13 imparted the opposite effects on these proteins, evidenced by the reduced expressions of p-p38 and p-ERK1/2 in H2O2-induced HUVECs with rA13 treatment, suggesting that ADAMTS13 could block p38/ERK signaling pathway in H2O2-induced HUVECs [Figure 4]. | Figure 4: A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) blocked p38/extracellular signal-regulated kinase (ERK) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were exposed to 0.5 mM H2O2 for 24 h with or without 1, 4 or 8 nM rA13 pretreatment for 30 min. The expressions of p38/ERK signaling pathway-related proteins were detected using western blot. Data from three independent replicates were presented as mean ± standard deviation. **P < 0.01 and ***P < 0.001.
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ADAMTS13 inhibited H2O2-induced injury of HUVECs via blocking p38/ERK signaling pathway
To further explore whether ADAMTS13 alleviates H2O2-induced injury of HUVECs by regulating p38/ERK signaling pathway, a p38 agonist p79350 was used to pretreat HUVECs for 1 h and then treated with or without rA13 at concentration of 8 nM for 30 min before H2O2 treatment. The expressions of p38/ERK signaling pathway-related proteins in H2O2-induced HUVECs with rA13 treatment were detected using western blot. As shown in [Figure 5]a, p79350 remarkably upregulated p-p38 and p-ERK1/2 expression when compared to the H2O2 + rA13 group. Then, results of CKK-8 assay indicated that the reduced viability of H2O2-induced HUVECs was partially revived by rA13 treatment compared with the H2O2 group, which was decreased by p79350 treatment [Figure 5]b. In addition, the enhanced apoptosis in HUVECs due to H2O2 induction was inhibited by rA13 treatment when compared with that in the H2O2 group, while p79350 exhibited the opposite impact on it, testified by the promoted apoptosis in the p79350 + H2O2 + rA13 group [Figure 5]c and [Figure 5]d. Results obtained from ELISA showed that rA13 treatment elevated the expression of PGF1α and reduced the expression of ET-1 in H2O2-induced HUVECs by contrast with the H2O2 group, which were then reversed by p79350 administration [Figure 5]e and [Figure 5]f. Collectively, rA13 was testified to inhibit H2O2-induced injury in HUVECs via blocking p38/ERK signaling pathway. | Figure 5: A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) inhibited hydrogen peroxide (H2O2)-induced injury in HUVECs via blocking p38/extracellular signal-regulated kinase (ERK) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were exposed to 0.5 mM H2O2 for 24 h. p79350 was used to pre-treat HUVECs for 1 h and then treated with 8 nM rA13 for 30 min before H2O2 treatment. (a) The expressions of p38/ERK signaling pathway-related proteins were detected using western blot. (b) The viability of HUVECs was detected using cell counting kit-8 assay. (c) The apoptosis of HUVECs was detected using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). (d) Quantification of apoptotic cells in TUNEL staining. (e) The expression of PGF1α in H2O2-induced HUVECs with rA13 treatment was detected using enzyme-linked immunosorbent assay (ELISA). (f) The expression of ET-1 in H2O2-induced HUVECs with rA13 treatment was detected using ELISA. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001.
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ADAMTS13 inhibited oxidative stress in H2O2-induced HUVECs via blocking p38/ERK signaling pathway
Relative to the H2O2 group, rA13 treatment decreased the level of ROS in H2O2-induced HUVECs while p79350 treatment exhibited the opposite effects, evidenced by the elevated level of ROS in p79350 + H2O2 + rA13 group [Figure 6]a. In addition, H2O2 induction elevated the protein expressions of Nox2 and Nox4 in HUVECs by contrast with the control group [Figure 6]b. Relative to the H2O2 + rA13 group, p79350 treatment partially elevated the protein expressions of Nox2 and Nox4 in H2O2-induced HUVECs with rA13 treatment, indicating that ADAMTS13 inhibited oxidative stress in H2O2-induced HUVECs through blocking p38/ERK signaling pathway. | Figure 6: A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) inhibited oxidative stress in hydrogen peroxide (H2O2) induced human umbilical vein endothelial cells (HUVECs) via blocking p38/extracellular signal-regulated kinase (ERK) signaling pathway. HUVECs were exposed to 0.5 mM H2O2 for 24 h. p79350 was used to pre-treat HUVECs for 1 h and then treated with 8 nM rA13 for 30 min before H2O2 treatment. (a) The level of ROS was detected using ROS assay kit. (b) The expressions of Nox2 and Nox4 were detected using western blot. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05 and ***P < 0.001.
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| Discussion | | |
Being a venous thromboembolic disease, DVT is characterized by high incidence, high mortality rate, and severe sequelae.[28] H2O2 is widely applied to induce HUVECs to establish DVT model.[17],[21] In view of this, 0.5 mM H2O2 was used to induce HUVECs in this study. It was reported that endothelial cell injury is a pivotal contributor to the development of DVT.[29] Many studies have substantiated that HUVECs were damaged by H2O2.[30],[31] In the present study, it was found that the viability of HUVECs was greatly decreased following the induction of H2O2. ADAMTS13, which is also known as von Willebrand factor-cleaving protease, could alleviate HUVECs injury in many diseases. For example, tr-ADAMTS13 increased the proliferation of VEGF-induced HUVECs.[32] The results obtained from CCK-8 assay in this study revealed that the decreased viability of HUVECs due to H2O2 induction was partially revived by treatment with rA13, indicating that ADAMTS13 could alleviate HUVECs injury induced by H2O2.
Previous studies have revealed that excessive oxidative stress is a prevalent contributor to vascular endothelial cell injury.[21],[33] In addition, it was evidenced that oxidative stress was high in patients suffering from DVT, implying that oxidative stress might act as a critical player in the pathology of venous thrombosis.[34] Considering this, it can be concluded that the intervention of oxidative stress may be an effective method to treat DVT. Besides, the inhibitory effects of ADAMTS13 on oxidative stress have been widely clarified. A case of recent study has claimed that ADAMTS13 inhibited oxidative stress in ischemia/reperfusion-induced acute kidney injury.[35] Zhou and co-workers held the opinion that ADAMTS13 suppressed oxidative stress to alleviate chronic kidney disease.[36] It is acknowledged that oxidative stress is caused by the excessive production of ROS.[37] In the present study, it was discovered that the increased ROS level in H2O2-induced HUVECs was then reduced by ADAMTS13 treatment. In addition, the protein contents of Nox2 and Nox4 in H2O2-induced HUVECs detected by western blot were inhibited following the treatment with rA13, suggesting the inhibitory effects of ADAMTS13 on oxidative stress in DVT.
It was reported that ROS promoted apoptosis in vascular endothelial cells and reduced the expressions of anti-apoptotic proteins.[38] A case of previous study has clarified that the apoptosis of vascular endothelial cells is the primary cause of venous thrombosis.[39] The inhibition of apoptosis was supposed to be an effective approach to alleviate DVT. For instance, the suppression of apoptosis by miR-195-5p could alleviate DVT.[40] Coagulation factor XII (FXII) was proved to protect against DVT through the regulation of apoptosis.[41] In the present study, the promoted apoptosis in HUVECs resulting from H2O2 induction was reduced by ADAMTS13 treatment.
Previous studies have demonstrated that ET-1 might participate in the formation of micro thrombosis.[42] The increase of ET-1 expression was considered as a thrombosis-related factor as well in DVT.[33] In this study, the expressions of PGF1α and ET-1 were also assessed and the results showed that H2O2 induction reduced PGF1α expression and elevated ET-1 expression, implying that H2O2 induction could facilitate thrombosis in HUVECs. Nevertheless, the decreased PGF1α expression and increased ET-1 expression in H2O2-induced HUVECs were reversed by treatment with rA13.
It was evidenced that the inhibition of ERK signaling pathway by ruscogenin could alleviate DVT.[43] In addition, Lou and co-workers have claimed that the inhibition of p-p38 and p-ERK could protect HUVECs against H2O2-induced oxidative stress and apoptosis.[17] More importantly, the phosphorylation of p38/ERK could be downregulated by rhADAMTS13, indicating that rhADAMTS13 could block p38/ERK signaling pathway.[16] In the present study, western blot demonstrated that H2O2 induction elevated the protein expressions of p-p38 and p-ERK1/2 in HUVECs, which were then reduced by treatment with rA13, implying that ADAMTS13 blocked p38/ERK signaling pathway in DVT. To further investigate the reaction mechanism of p38/ERK signaling pathway in DVT, p79350, an agonist of p38, was used to pretreat HUVECs and above functional experiments were conducted again. The results revealed that the inhibitory effects of ADAMTS13 on the viability damage, apoptosis and oxidative stress in H2O2-induced HUVECs were partially counteracted by p79350 pretreatment, which suggested that ADAMTS13 inhibited HUVECs injury and oxidative stress induced by H2O2 through the inactivation of p38/ERK signaling pathway.
| Conclusion | | |
To conclude, this study unmasked the functional role of ADAMTS13 in H2O2-induced HUVECs injury and oxidative stress and identified that ADAMTS13 could block p38/ERK signaling pathway in H2O2-induced HUVECs, which for the first time revealed the mechanism by ADAMTS13 exerted protective effects on DVT.
Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Financial support and sponsorship
This work was supported by the Youth project of Suzhou TCM Hospital (No. YQN2021004), Science and Technology Development Project of Suzhou City of China (No. SKY2021011), Science and Technology Development Project of Suzhou City of China (No. SKJYD2021125), Special Project on Diagnosis and Treatment of Key Clinical Diseases in Suzhou (No. LCZX202214), Scientific and Technological Project in Suzhou (No. SLJ2022024) and Youth Science and Technology Project of “Promoting Health through Science and Education” in Suzhou (No. KIW2022042).
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Jianjie Rong,
Department of Vascular Surgery, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, No. 18, Yangsu Road, Suzhou 215009, Jiangsu
China
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/cjop.CJOP-D-23-00101
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6] |
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