PTP1B inhibition ameliorates inflammatory injury and dysfunction in ox-LDL-induced HUVECs by activating the AMPK/SIRT1 signaling pathway via negative regulation of KLF2

Atherosclerosis is a key pathogenic factor of cardiovascular diseases. However, the role of protein tyrosine phosphatase 1B (PTP1B) in oxidized low-density lipoprotein (ox-LDL)-treated vascular endothelial cells remains unclear. The aim of the present study was to explore the possible physiological roles and mechanism of PTP1B in atherosclerosis using HUVECs as an in vitro model. PTP1B expression was assessed by reverse transcription-quantitative PCR. Cell viability was measured using the Cell Counting Kit-8 and lactate dehydrogenase activity assays. Levels of inflammatory factors, including IL-1β, IL-6 and TNF-α, and oxidative stress factors, including malondialdehyde, superoxide dismutase and glutathione peroxidase, were assessed using ELISA and commercially available kits, respectively. Furthermore, TUNEL assay and western blotting were performed to assess the extent of apoptosis-related factors, including Bcl-2, Bax, Cleaved caspase-3 and Caspase-3. Tube formation assay was used to assess tubule formation ability and western blotting was to analyze VEGFA protein level. Binding sites for the transcription factor Kruppel-like factor 2 (KLF2) on the PTP1B promoter were predicted using the JASPAR database and verified using luciferase reporter assays and chromatin immunoprecipitation. The protein levels of phosphorylated 5'AMP-activated protein kinase (p-AMPK), AMPK and SIRT1 were measured using western blotting. The results demonstrated that the PTP1B mRNA and protein expression levels were significantly upregulated in oxidized low-density lipoprotein (ox-LDL)-induced HUVECs. In addition, ox-LDL-induced HUVECs transfected with short hairpin RNA against PTP1B exhibited a significant increase in cell viability, reduced inflammatory factor levels, apoptosis and oxidative stress, as well as increased tubule formation ability. KLF2 was found to negatively regulate the transcriptional activity of PTP1B. KLF2 knockdown reversed the protective effects of PTP1B knockdown on ox-LDL-induced HUVECs. KLF2 knockdown also abolished PTP1B knockdown-triggered AMPK/SIRT1 signaling pathway activation in ox-LDL-induced HUVECs. To conclude, the results of the present study suggested that PTP1B knockdown can prevent ox-LDL-induced inflammatory injury and dysfunction in HUVECs, which is regulated at least in part by the AMPK/SIRT1 signaling pathway through KLF2.


Introduction
Endothelial cells (ECs) serve to maintain vascular integrity and homeostasis by sensing and responding to pathological and physiological stimuli (1)(2)(3)(4). During the onset of vascular disease, major phenotypical alterations occur in the ECs, resulting in increased vascular permeability, release of large quantities of inflammatory cytokines (IL-8, IL-6 and IL-1β) and leukocyte adhesion (5)(6)(7). These pathological changes in the blood vessel wall structure and function in turn increases the risk of atherosclerosis (8)(9)(10). It has previously been reported that EC inflammation and apoptosis serve key roles in the initiation and development of atherosclerosis, hypertension, diabetes and other cardiovascular diseases (11). Furthermore, endothelial dysfunction is regarded to be one of the first stages in the pathophysiology of atherosclerosis (12). Loss of morphological and functional integrity in vascular ECs has been reported to be attributed to inflammation and apoptosis (13). Therefore, therapeutic strategies targeting inflammation, apoptosis and vascular EC dysfunction may be important for the treatment of atherosclerosis.
Protein tyrosine phosphatase 1B (P TP1B) is a non-transmembrane protein tyrosine phosphatase that has been documented to be a negative regulator in diabetes and obesity signaling (14)(15)(16). In addition, it has reported roles in the malignant transformation of various cancers, including pancreatic cancer and resistance in cancer treatments, such as dendritic cell-based cancer immunotherapy (14)(15)(16). Accumulating evidence indicates that PTP1B is also involved in atherosclerosis (17). Thompson et al (18) previously reported that PTP1B inhibitors can prevent and reverse atherosclerotic plaque formation in low-density lipoprotein (LDL) receptor -/mice with atherosclerosis, thereby reducing the risk of cardiovascular disease. Improved glucose homeostasis, reduced circulating lipids and atherosclerotic plaque lesions, have also been observed in myeloid-PTP1B-knockout mice (apolipoprotein E -/-/lysozyme M-PTP1B) with atherosclerosis (19). In addition, endothelial PTP1B has been reported to serve as the main regulator of EC proliferation in cardiovascular disease (20). By contrast, PTP1B depletion was found to induce endothelium-dependent vasorelaxation in microvessels in animal models of heart failure and diabetes (21,22). However, to the best of our knowledge, the potential effects of PTP1B on the inflammation, apoptosis and dysfunction of oxidized (ox)-LDL-induced vascular ECs and associated mechanisms remain unreported.
Therefore, the present study aimed to investigate the possible role of PTP1B in ox-LDL-treated HUVECs. The study reported the efficacy of PTP1B on the proliferation, inflammation, apoptosis, oxidative stress, tubule formation, Kruppel-like factor 2 (KLF2) and 5'AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signaling pathway in ox-LDL-induced HUVECs through functional experiments and mechanism assays.

Materials and methods
Cell culture. Immortalized HUVECs were purchased from Shanghai EK-Bioscience Biotechnology Co., Ltd. (cat. no. CC-Y1285). Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Hyclone; Cytiva) and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO 2 at 37˚C. To establish an in vitro atherosclerosis model, HUVECs were treated with 100 µg/ml ox-LDL (Guangzhou Yiyuan Biological Technology Co., Ltd.) for 24 h at 37˚C. Untreated cells were regarded as the control group.
Cell Counting Kit (CCK)-8 assay. HUVECs (4x10 4 cells/well) seeded into a 96-well plate were transfected with sh-PTP1B in the presence or absence of sh-KLF2 before treatment with ox-LDL (100 µg/ml) for 24 h at 37C (23)(24)(25). Subsequently, 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added to each well and the plates were incubated for 2 h at 37˚C. The optical density values were analyzed using a Thermo Multiskan FC microplate reader (Thermo Fisher Scientific, Inc.) at 450 nm.
Lactate dehydrogenase (LDH) activity assay. HUVECs plated at a density 1x10 4 cells/well in 96-well plates were transfected with sh-PTP1B in the presence or absence of sh-KLF2 and underwent ox-LDL treatment (100 µg/ml) for 24 h at 37˚C. Cells were subsequently harvested from the culture plate and the LDH activity levels were detected by LDH activity kit (cat. no. A020; Nanjing Jiancheng Bioengineering Institute) at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).
ELISA. Briefly, HUVECs were seeded into 96-well plates (5x10 3 cells/well). Following the aforementioned treatment, cell supernatant was collected after centrifugation at 2,000 x g for 5 min at 4˚C. IL-6 (cat. no. ab178013), IL-1β (cat. no. ab214025) and TNF-α (cat. no. ab181421) levels in the culture supernatant were determined using ELISA kits from Abcam according to the manufacturer's protocols. The absorbance was determined at 450 nm using an xMark microplate absorbance spectrophotometer (Bio-Rad Laboratories, Inc.).
TUNEL assay. Cell apoptosis was detected using an In Situ Cell Death Detection Kit (cat. no. 11684817910; Roche Diagnostics GmbH). Cells were fixed with 4% paraformaldehyde at room temperature away from light for 30 min and then incubated with proteinase K for 15 min in 37˚C. Subsequently, cells were placed in 3% H 2 O 2 for 15 min at room temperature to inhibit endogenous peroxide. HUVECs were then stained with the TUNEL detection kit at 37˚C for 60 min and co-labeled with the DAPI working solution (1 µg/ml) for 10 min at 37˚C according to the manufacturer's protocols. Labeled HUVECs were washed with PBS buffer. Next, cells on slips were mounted using DAPI-containing mounting medium (Vector Laboratories, Inc.) and visualized using a fluorescence microscope (Nikon Eclipse 80i; Nikon Corporation), and >10 fields per section for each sample were examined. The TUNEL positive cell rate (%) was calculated using the software of Developer XD 1.2 (Definiens AG) according to the following formula: (Number of positive cells/total number of cells) x100.
Endothelial tube formation assay. HUVECs were seeded at a density of 5x10 3 cells/well into 96-well plates pre-coated with 50 µl/well Matrigel (Corning, Inc.) at 37˚C for 2 h. HUVECs were seeded into 96-well plates and transfected with sh-PTP1B in the presence or absence of sh-KLF2 and underwent ox-LDL treatment (100 µg/ml). Following incubation for 24 h at 37˚C, tubules characterized by the capillary-like structures were imaged using a light microscope in five randomly selected fields (magnification, x40).
Dual-luciferase reporter assay. Briefly, the potential interaction between KLF2 and the PTP1B promoter were predicted using data from the 9th release (2022) of JASPAR database (http://jaspar.genereg.net). The wild-type and mutant PTP1B promoter fragments, including predicted KLF2 sites, were amplified and cloned downstream of the luciferase reporter gene in the firefly luciferase reporter pGL3 vector (Promega Corporation). HUVECs were transfected with 2.5 µg Ov-KLF2 or 2.5 µg Ov-NC plasmids, with 100 ng of luciferase reporter plasmids driven by wild-type or mutant PTP1B promoter, using Lipofectamine 2000 at 37˚C for 48 h before luciferase activity was detected using the Dual-Luciferase Reporter Assay Kit (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.
Chromatin immunoprecipitation (ChIP). ChIP experiments were performed using the ChIP-IT kit (cat. no. 53008; Active Motif, Inc.) according to the manufacturer's instructions as previously described (27). Briefly, HUVECs were cross-linked with 1% formaldehyde at 37˚C for 10 min. The cell lysates were sonicated using a 10 sec on and 10 sec off mode for 12 cycles on ice. Following quenching with 2.5 M glycine for 5 min at room temperature, the supernatant was collected, added to 60 µl Protein A Agarose beads (cat. no. 9863; Cell Signaling Technology, Inc.) and mixed for 1 h after centrifugation. DNA was immunoprecipitated from the 100 µl cell lysates using 2 µg KLF2 antibody (cat. no. MBS9211982; MyBioSource) for a 2-h incubation at 4˚C. The beads were washed using a magnetic separation rack and the bound chromatin was eluted in ChIP Elution Buffer with Proteinase K mixer. PCR amplification of the PTP1B binding site was then performed using the precipitated DNA by means of SYBR Premix Ex Taq™ II kit (cat. no. RR420A; Takara Bio, Inc.). The following thermocycling conditions were used: 95˚C for 7 min, followed by 45 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 15 sec and extension at 72˚C for 30 sec, with a final extension at 60˚C for 1 min followed by cooling at 40˚C for 5 min. Next, the immunoprecipitated DNA was purified using a ChIP DNA purification kit (cat. no. D0033; Beyotime Institute of Biotechnology). Nonspecific antibody against IgG (2 µg; 1:40; cat. no. sc-2025; Santa Cruz Biotechnology, Inc.) served as a negative control.
Statistical analysis. All statistical analysis was performed using GraphPad Prism software (version 5.01; GraphPad Software, Inc.). All data are presented as the mean ± SD from at least three independent experiments. Statistical comparisons between two groups were performed using unpaired Student's t-test, whereas comparisons among >2 groups was performed using one-way ANOVA followed by Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

PTP1B knockdown restores cell viability in ox-LDL-induced
HUVECs. To investigate the role of PTP1B in atherosclerosis, the expression of PTP1B in HUVECs was measured. PTP1B mRNA and protein expression levels were significantly increased in ox-LDL-induced cells compared with those in the control group ( Fig. 1A and B). Subsequently, sh-PTP1B constructs were transfected into HUVECs to knock down PTP1B expression. The results from the RT-qPCR and western blotting experiments demonstrated that the PTP1B mRNA and protein expression levels were significantly reduced following transfection with sh-PTP1B#1 or shPTP1B#2 ( Fig. 1C and D). Since sh-PTP1B#2 demonstrated a greater transfection efficiency, it was selected for subsequent experiments ( Fig. 1C and D). Cell viability was then assessed using the CCK-8 assay. The results demonstrated that ox-LDL significantly reduced the cell viability of HUVECs, which was in turn reversed by PTP1B silencing (Fig. 1E). Furthermore, significantly increased LDH activity was observed in ox-LDL-treated cells compared with the control group, which was also reversed by sh-PTP1B transfection in response to ox-LDL (Fig. 1F).

PTP1B knockdown alleviates ox-LDL-induced inflammatory damage in HUVECs.
To evaluate the effects of PTP1B silencing on inflammatory injury in HUVECs in response to ox-LDL, parameters of inflammation and cell apoptosis were measured. The results demonstrated that ox-LDL treatment significantly enhanced IL-6, IL-1β and TNF-α levels compared with those in the control group ( Fig. 2A). However, sh-PTP1B transfection counteracted these effects of ox-LDL on the three inflammatory factors aforementioned (Fig. 2A). Furthermore, cell apoptosis was found to be significantly increased by ox-LDL, which was reversed following the knockdown of PTP1B expression ( Fig. 2B and C). Western blotting results demonstrated a significant reduction in the Bcl-2 protein expression levels and a significant increase in Bax and cleaved caspase-3 protein expression levels in ox-LDL-treated cells compared with those in the control group (Fig. 2D). However, the effects of ox-LDL on the expression of these aforementioned proteins associated with apoptosis were reversed by PTP1B knockdown (Fig. 2D).

PTP1B knockdown suppresses ox-LDL-induced oxidative stress in HUVECs and restores tubule-formation ability.
Subsequently, the role of PTP1B in oxidative stress in HUVECs induced by ox-LDL was explored. The results demonstrated that ox-LDL treatment significantly increased MDA levels whilst significantly decreasing SOD and GSH-Px activity compared with those in the control group (Fig. 3A). By contrast, PTP1B knockdown significantly reversed the increase in MDA levels whilst also reversing the reduction in SOD and GSH-Px activity following ox-LDL treatment (Fig. 3A). The effects of PTP1B on the tubule-formation ability of HUVECs were investigated. Ox-LDL significantly reduced the number of tubules compared with that in the control group, which was also significantly reversed by PTP1B knockdown ( Fig. 3C and D). Furthermore, western blotting demonstrated that ox-LDL significantly reduced VEGFA expression in HUVECs compared with that in control cells, while a significant increase in VEGFA protein expression was observed in ox-LDL-treated HUVECs following PTP1B knockdown compared with that in cells treated with ox-LDL alone (Fig. 3D).
KLF2 negatively regulates PTP1B transcription. Using the JASPAR database, the transcription factor KLF2, was predicted to bind to the PTP1B promoter (Fig. 4A). KLF2 mRNA and protein expression levels were both found to be significantly reduced in HUVECs following treatment with ox-LDL ( Fig. 4B and C). To explore the effects of KLF2 on PTP1B in HUVECs, KLF2 was overexpressed and transfection efficiency was verified, as evidenced by the significantly increased KLF2 expression in cells transfected with the Ov-KLF2 plasmid compared with that in cells transfected with the Ov-NC plasmid ( Fig. 4D and E). It was demonstrated that the luciferase activity of the wild-type PTP1B promoter was significantly inhibited by KLF2 overexpression, whereas the mutant PTP1B promoter group displayed no changes in the luciferase activity (Fig. 4F). ChIP was performed to further verify the potential binding of KLF2 on the PTP1B promoter. The results demonstrated that the PTP1B DNA sequence was significantly enriched in the KLF2 group compared with that in the IgG group (Fig. 4G). Furthermore, KLF2 overexpression significantly inhibited the expression of both PTP1B mRNA and protein compared with that in cells transfected with the Ov-NC plasmid (Fig. 4H and I). This suggested a negative regulatory mechanism exerted by KLF2 against PTP1B expression in HUVECs.

KLF2 knockdown reverses the protective effects of PTP1B
silencing on ox-LDL-induced HUVECs by regulating the AMPK/SIRT1 signaling pathway. To investigate the role of KLF2 in ox-LDL-treated HUVECs with PTP1B expression Figure 3. Knockdown of PTP1B expression alleviates ox-LDL-induced oxidative stress in HUVECs to restore tubule-formation ability. (A) Levels of MDA, SOD and GSH-Px were evaluated using corresponding kits. (B) Tubule-formation ability was explored using endothelial tubule formation assay and (C) quantified. Magnification, x40. (D) VEGFA protein expression was measured using western blotting. Results represent the mean ± SD. ** P<0.01 and *** P<0.001. MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; PTP1B, protein tyrosine phosphatase 1B; ox-LDL, oxidized low-density lipoprotein; sh, short-hairpin; VEGFA, vascular endothelial growth factor A; NC, negative control. knocked down, sh-KLF2 was transfected into HUVECs and KLF2 expression was prominently decreased after transfection of sh-KLF2#1/2 plasmids, which verified the transfection efficiency using RT-qPCR and western blotting ( Fig. 5A and B). Cells transfected with sh-KLF2#2 exhibited lower expression levels of KLF2 compared with those in the sh-KLF2#1 group (Fig. 5A and B). Therefore, sh-KLF2#2 was used for subsequent experiments. Compared with those in the sh-PTP1B-only group, co-transfection with sh-PTP1B and sh-KLF2 slightly reduced cell viability whilst significantly increasing LDH activity (Fig. 5C and D), indicating a reversal of the protective effects initially exerted by PTP1B knockdown. In addition, IL-6, IL-1β and TNF-α levels in ox-LDL-induced HUVECs were significantly elevated by the knockdown of KLF2 and PTP1B compared with those in cells with only PTP1B expression knocked down (Fig. 5E). KLF2 knockdown also significantly reversed the protective effects mediated by sh-PTP1B transfection against cell apoptosis (Fig. 5F), in addition to the expression levels of Bcl-2, Bax and cleaved caspase-3 (Fig. 6B). Furthermore, KLF2 knockdown significantly increased MDA levels whilst significantly decreasing SOD and GSH-Px activity compared with those in cells transfected with sh-PTP1B only (Fig. 5G).
The number of tubules formed and VEGFA expression were also significantly reduced in the sh-KLF2 + sh-PTP1B compared with those in the sh-PTP1B-only group ( Fig. 6A and B). The results of the western blotting assay demonstrated that ox-LDL significantly reduced the levels of AMPK phosphorylation and SIRT1 expression (Fig. 6C). However, sh-PTP1B transfection significantly reversed these effects of ox-LDL on the AMPK/SIRT1 signaling pathway (Fig. 6C). Furthermore, subsequent knockdown of both KLF2 and PTP1B significantly counteracted these regulatory effects of sh-PTP1B on AMPK phosphorylation and SIRT1 expression (Fig. 6C).

Discussion
In the present study, the role of PTP1B in ox-LDL-induced HUVECs was investigated. The results demonstrated that PTP1B knockdown significantly restored cell viability, inhibited inflammatory cell injury whilst also restoring tubule formation ability. Furthermore, the results demonstrated that PTP1B expression was negatively regulated by KLF2, which may be associated with the AMPK/SIRT1 signaling pathway.
Atherosclerosis is a chronic inflammatory disease that is caused by abnormal responses of the blood vessel wall to a number of noxious stimuli (28)(29)(30). Vascular EC injury is considered to be a common pathological cause of cardiovascular diseases (31). HUVECs have been widely acknowledged to be a useful model for research on the human vascular endothelium (32)(33)(34)(35). Although this model does not represent all EC types in an organism, it is nevertheless a viable model for studying the main molecular pathways and properties underlying endothelial functions (36). Ox-LDL can accelerate EC inflammation, apoptosis and endothelial-mesenchymal transition, which serves a key role in mediating the early stages of lesion formation during atherosclerosis (37). Ox-LDL also triggers lipid metabolism disorders, leading to EC injury and death (38). In the present study, ox-LDL stimulation was used to establish an in vitro atherosclerosis model, where a dose of ox-LDL (100 µg/ml) was selected based on previous studies (23)(24)(25). The results demonstrated that HUVECs treated with ox-LDL exhibited reduced cell viability, increased inflammatory factor levels, elevated apoptosis rates and reduced tubule formation capabilities, all of which are consistent with previous reports (23,24). PTP1B has been previously reported to serve a role in atherosclerosis and contribute to the pathophysiology of ECs (19,39). The present study demonstrated that PTP1B expression was upregulated in HUVECs after treatment with ox-LDL. Following PTP1B knockdown, HUVEC viability and tubule formation ability were significantly restored, whereas LDH activity, inflammatory factor levels and cell apoptosis were suppressed in cells stimulated with ox-LDL. Furthermore, the oxidative stress levels of the cells were also decreased following PTP1B knockdown. These findings were in consistency with previous reports, which showed that the upregulation of the combination of the cAMP response element-binding protein and lysine methyltransferase 5A inhibited the hyperglycemia-induced inflammatory factor levels by regulating PTP1B expression in vascular endothelial cells (40). These results suggested that PTP1B knockdown may protect HUVECs against inflammatory injury and dysfunction induced by ox-LDL.
binding site of KLF2 on the PTP1B promoter. The results demonstrated that KLF2 mRNA and protein expression levels were decreased in ox-LDL-treated cells compared with those in the control cells. To verify the binding between KLF2 and the PTP1B promoter, dual-luciferase reporter and ChIP assays were performed. The results demonstrated an interaction between KLF2 and the PTP1B promoter. KLFs belong to the zinc finger family of transcription factors that serve key roles in biological processes, including cell proliferation, inflammation and differentiation (43). KLF2 has been previously demonstrated to serve a role in the regulation of inflammatory activation in endothelial cells (44). A previous study reported that IFN regulatory factor 2-binding protein 2 can protect against ox-LDL-induced endothelial inflammation and epithelial-mesenchymal transition by activating KLF2 expression (45). In addition, Li et al (46) reported that synoviolin 1 overexpression inhibited the ox-LDL-induced apoptosis of endothelial cells, which was positively regulated by KLF2. Another previous study demonstrated that ox-LDL could reduce the levels of downstream regulators, such as myocyte enhancer factor 2C, where ERK5 ameliorated ox-LDL-induced EC death, inflammation and dysfunction by inhibiting the ERK5/myocyte enhancer factor 2C/KLF2 signaling pathway in an ox-LDL-induced primary human umbilical vein endothelial cell model (47). In agreement with these previous studies, the present study showed that KLF2 knockdown reversed the effects of PTP1B silencing on ox-LDL-induced HUVECs by inhibiting cell viability and endothelial function, whilst promoting inflammation, oxidative stress and apoptosis.
In conclusion, the present study demonstrated that PTP1B may serve a regulatory role in inflammation and endothelial dysfunction in ox-LDL-induced HUVECs. The results also suggested that PTP1B expression is negatively regulated by KLF2, which may be dependent on the AMPK/SIRT1 signaling pathway. The present study provided a potential novel therapeutic target for the treatment of endothelial dysfunction that occurs during atherosclerosis.