ABSTRACT
Background
Fibroblast growth factor receptor (FGFR)2 expression was decreased in hypertension patients while its role in hypertension was not explored. This experiment aimed to investigate the expression ofFGFR2 in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs) and the role of FGFR2 in improving AngII-induced hypertension-related endothelial dysfunction.
Methods
AngII-induced HUVECs simulated the hypertension model in vitro. The expression of FGFR2 in Ang II-induced HUVECs and transfected HUVECswas detected by RT-qPCR and western blot. The viability, apoptosis, migration and tube formation ability of Ang II-induced HUVECs were analyzed by Methyl Thiazolyl Tetrazolium (MTT) assay, flow cytometry analysis, wound healing assay and tube formation assay.Detectionof lactate dehydrogenase (LDH), caspase 3, Nitric Oxide (NO) and oxidative stress levels was conducted by assay kits and reactive oxygen species (ROS) level was detected by DCFH-DA assay. The expression of apoptosis-related proteins, protein kinase B(Akt)/nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway-related proteins, phospho(p)-endothelial nitric oxide synthase (eNOS) and eNOS was determined by western blot.
Results
The expression of FGFR2 was decreased in Ang II-induced HUVECs. FGFR2overexpression increased viability, suppressed apoptosis and oxidative stress, and improve endothelial dysfunction of AngII-induced HUVECs through activating the Akt/Nrf2/ARE signaling pathway. MK-2206 (Akt inhibitor) could weaken the effect of FGFR2overexpression to reduce viability, promote apoptosis and oxidative stress, and aggravate endothelial dysfunction of Ang II-inducedHUVECs.
Conclusion
Inconclusion, FGFR2activated the Akt/Nrf2/ARE signaling pathway to improve AngII-induced hypertension-related endothelial dysfunction.
Introduction
Hypertension is one of the main risk factors of cardiovascular disease. According to the Chinese hypertension survey, the prevalence rate of adult hypertension in China is 27.9%, showing an increasing trend and the awareness rate, treatment rate and control rate are respectively 51.6%, 45.8% and 16.8% (Citation1). Compared with the previous survey, although the overall level has been improved, its control rate is still relatively low (Citation1). Stroke and ischemic heart disease caused by hypertension are the leading causes of death worldwide, a major global health challenge, and significantly increase the economic burden on society (Citation2). Therefore, it is very important to reduce the socio-economic burden on our country by enhancing hypertension prevention and control to reduce the occurrence and development of cardiac and cerebrovascular disease.
Fibroblast growth factor receptor (FGFR)2 is a member of the receptor tyrosine kinase subfamily, which also includes FGFR1, FGFR3 and FGFR4 (Citation3). Serum FGFR2 was significantly reduced in fracture patients, and FGFR2 could regulate osteoblast viability and apoptosis (Citation4). FGFR2 deletion led to defective vesical urethral epithelial regeneration after cyclophosphamide injury (Citation5). FGFR2 expression was downregulated in caerulein-induced pancreatic cells and FGFR2 overexpression decreased the caerulein-induced inflammatory injury in pancreatic cells (Citation6). FGFR2 deletion in tubular cells exacerbated acute renal dysfunction and tubular cell apoptosis induced by ischemia/reperfusion or cisplatin (Citation7). It can be seen from the above that the decrease of FGFR2 is harmful to the body, and the increase of FGFR2 expression can reverse the body injury. Peripheral blood microarray sequencing in patients with essential hypertension and volunteers with normal blood pressure in the GSE24752 dataset showed down-regulated FGFR2 expression in hypertension patients (Citation8). However, its role in hypertension has not been reported.
Hypertension itself is often accompanied by vascular endothelial dysfunction, and hypertension can also cause vascular endothelial dysfunction. There are two main mechanisms involved. First, increased blood pressure leads to increased superoxide production, endothelial nitric oxide synthase (eNOS) decoupling, and decreased nitric oxide bioavailability (Citation9). Second, angiotensin II (AngII) reduces eNOS activity and nitric oxide (NO) bioavailability to promote the occurrence of vascular endothelial dysfunction (Citation10). A previous study established the AngII-induced HUVECs model to simulate the hypertension in vitro (Citation11) and this study also conducted basing on the AngII-induced HUVECs model.
Therefore, this experiment aimed to investigate the expression of FGFR2 in AngII-induced HUVECs and the role of FGFR2 in improving AngII-induced hypertension-related endothelial dysfunction.
Material and methods
Cell culture and treatment
Human umbilical vein endothelial cells (HUVECs; cat. no. PCS‑100‑010) were provided by the American Type Culture Collection and cultured in DMEM added with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2.
Overexpression (Oe)-FGFR2 and the empty plasmid were purchased from Shanghai GenePharma Co., Ltd. Cells were incubated in 6‑well plates (3×105 cells/well) for 24 h at 37°C and transfected with Oe-FGFR2 and Oe-NC using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.). The transfection efficiency was confirmed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) 48 h following transfection.
After cell transfection for 48 h, HUVECs were induced by 1 µM AngII (SigmaAldrich, Merck, Germany) for 24 h for the experiment. To block protein kinase B (Akt), cells were incubated with 5 µM of Akt inhibitor MK-2206 (MCE, NJ, USA) for 0.5 h prior to AngII treatment.
RT‑qPCR
Following treatment, total RNA was extracted from HUVECs by TRIzol® reagents (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription was conducted using a PrimeScript RT Reagent kit (Takara Bio, Inc.) to reverse transcribe the RNA into complementary DNA (cDNA). qPCR reaction was performed using Kapa SYB® FAST qPCR Master Mix and ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression level of FGFR2 was quantified using the 2‑∆∆cq method and normalized to GAPDH (Citation12). The sequences of the primers were as follows: FGFR2 forward, 5”-AGCACCATACTGGACCAACAC-3,‘ reverse, 5’- GGCAGCGAAACTTGACAGTG-3;‘ and GAPDH forward, 5’- AATGGGCAGCCGTTAGGAAA-3‘ and reverse, 5’- GCGCCCAATACGACCAAATC-3.”
Western blot
Following treatment, HUVECs were lysed with RIPA lysate (Beyotime) and centrifuged at 4°C for 10 min with 2000 × g to obtain the proteins, which was quantified by the BCA assay kit (Beyotime). Proteins (30 µg/lane) were separated by 10% sodium dodecyl sulfate‑polyacrylamide gel (SDS‑PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% skimmed milk at room temperature for 1 h, membranes were incubated with primary antibodies against FGFR2 (ab109372; 1/1000; Abcam), B-cell lymphoma-2 (Bcl-2; ab32124; 1/1000; Abcam), Bcl-2-associated X protein (Bax; cat. no. ab32503; 1/1000; Abcam), cleaved poly(ADP-ribose) polymerase (PARP) (cat. no. ab32064; 1/1000; Abcam), PARP (cat. no. ab191217; 1/1000; Abcam), phospho (p)-eNOS (cat. no. ab215717; 1/1000; Abcam), eNOS (cat. no. ab300071; 1/1000; Abcam), p-Akt (cat. no. ab38449; 1/1000; Abcam), Akt (cat. no. 10176–2-AP; 1/1000; Proteintech), nuclear factor E2-related factor 2 (Nrf2; cat. no. ab62352; 1/1000; Abcam), NADPH quinone oxidoreductase-1 (NQO-1; cat. no. ab80588; 1/10000; Abcam), heme oxygenase-1 (HO-1; cat. no. ab52947; 1/2000; Abcam) and glyceraldehyde-phosphate dehydrogenase (GAPDH; cat. no. ab9485; 1/2500; Abcam) overnight at 4°C. Subsequently, the membranes were incubated with secondary antibody against goat anti‑rabbit IgG H&L (cat. no. ab6721; 1/2000; Abcam) for 2 h at room temperature. The protein bands were visualized using enhanced chemiluminescence reagents (Beyotime) and bands intensity were quantified by ImageJ software (version 1.46; National Institutes of Health). Thereafter, membranes were stripped with Western blotting stripping buffer (Takara Bio, Inc.) and then washed and re-probed with the aforementioned antibodies. GAPDH was used as an endogenous loading control.
Methyl Thiazolyl Tetrazolium (MTT) assay
HUVECs were seeded into a 96-well cell culture plate (5,000 cells/well) and cultured at 37°C for 24 h. Following treatment, each well was added with 10 µl MTT solution (5 mg/ml; Beyotime) and continuously incubated for 4 h. After removing the supernatant, the blue-purple crystals were dissolved using 200 µl DMSO in dark for 15 min, which was detected by a microplate reader to record the absorbance at 490 nm.
Detection of lactate dehydrogenase (LDH), caspase 3, NO and oxidative stress levels
Following treatment, HUVECs were centrifuged to obtain the supernatant. The LDH release, caspase 3 activity and levels of NO, catalase (CAT), glutathione peroxidase (GSH-Px) and malondiadehyde (MDA) in the supernatant were in turn detected by LDH assay kit (cat. no. A020-2-2; Nanjing Jiancheng Bioengineering Institute), caspase 3 activity assay kit (cat. no. C1115; Beyotime), NO assay kit (cat. no. A013-2-1; Nanjing Jiancheng Bioengineering Institute), CAT assay kit (cat. no. A007-1-1; Nanjing Jiancheng Bioengineering Institute), GSH-Px assay kit (cat. no. A005-1-1; Nanjing Jiancheng Bioengineering Institute), MDA assay kit (cat. no. A003-4-1; Nanjing Jiancheng Bioengineering Institute).
Flow cytometry analysis
Following treatment, the apoptosis of HUVECs was detected by transporting phosphatidylserine with an FITC Annexin V apoptosis kit (BD Biosciences). Briefly, HUVEC cells were incubated with propidium and Annexin V-FITC in dark for 10 min and cell apoptosis was analyzed by a flow cytometer (BD Biosciences).
Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay
2“,7”-dichlorodihydrofluorescein-diacetate (DCFH-DA; Beyotime) was diluted by serum‑free medium to the final concentration of 10 µmol/l. Following treatment of HUVECs, the cell culture medium was discarded and HUVECs were incubated with enough diluted DCFH‑DA for 15 min at 37°C. After removing the DCFH‑DA, HUVECs were washed by serum‑free medium and the absorbance was detected at 525 nm emission wavelength to calculate the fluorescence intensity. The relative fluorescence intensity was analyzed by using ImageJ software (version 1.46; National Institutes of Health).
Wound healing assay
HUVECs were seeded in six‑well plates and cultured until the cell confluence reached>90%. A sterile 10-μl tip was used to make a homogenous wound and the scratched cells were cultured in serum‑free medium for 24 h. The widths of the scratch at 0 and 24 h were observed and photographed using an inverted light microscope.
Tube formation assay
Following treatment, HUVECs were incubated at 37°C for 24 h and tubes characterized by the capillary-like structures were observed and photographed using a light microscope.
Statistical analysis
Data are presented as the mean ± standard deviation (SD) of three independent experiments and three cell replicates were performed in each experiment. Data are analyzed by statistical analysis using GraphPad Prism software (version 1.8.0; GraphPad Software, inc.). The unpaired Student’s t‑test was used to compare differences between two groups and one‑way ANOVA followed by Turkey’s post hoc test was used to compare differences among multiple groups. P < .05 was considered statistically significant.
Results
Overexpression of FGFR2 increased the viability of Ang II-induced HUVECs
FGFR2 expression has been uncovered to be reduced in hypertension patients. To clarify the specific role of FGFR2 in hypertension, FGFR2 expression was examined in Ang II-treated HUVECs. Following Ang II treatment, it was discovered that both the mRNA level () and protein level () of FGFR2 were decreased in Ang II-induced HUVECs. To determine the impacts of FGFR2 on the Ang II-treated HUVECs, FGFR2 was overexpressed following transfection of FGFR2 overexpression plasmids. RT-qPCR and western blot analysis manifested that both the mRNA level () and protein level () of FGFR2 were upregulated in HUVECs transfected with Oe-FGFR2. Subsequently, Control group, Oe-NC group and Oe-FGFR2 group were divided. Through MTT assay, HUVECs viability was evaluated. As depicted in , the viability of HUVECs was decreased by Ang II induction. FGFR2 overexpression increased the viability of Ang II-induced HUVECs. In addition, LDH release is a good indicator of cellular damage. The results from LDH assay revealed that the raised LDH activity in HUVECs exposed to Ang II was declined after FGFR2 was overexpressed (). Conclusively, FGFR2 elevation might alleviate Ang II-induced HUVECs viability loss.
Figure 1. FGFR2 expression was decreased in Ang II-induced HUVECs. (A) the mRNA expression of FGFR2 in Ang II-induced HUVECs was detected by RT-Qpcr. (B) the protein expression of FGFR2 in Ang II-induced HUVECs was detected by western blot. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group.
Figure 2. Overexpression of FGFR2 increased the viability of Ang II-induced HUVECs. (A) the mRNA expression of FGFR2 in HUVECs transfected with Oe-FGFR2 was detected by RT-Qpcr. (B) the protein expression of FGFR2 in HUVECs transfected with Oe-FGFR2 was detected by western blot. (C) the viability of Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by MTT assay. (D) the LDH release of Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by LDH assay kit. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group. ##P<.01 and ###P<.001 vs. Ang II + Oe-NC group.
Overexpression of FGFR2 inhibited Ang II-induced apoptosis in HUVECs
Furthermore, to uncover the effects of FGFR2 on the apoptosis of Ang II-induced HUVECs, flow cytometry analysis was applied. On the contrary, it turned out that the apoptosis of HUVECs was increased by Ang II induction and FGFR2 overexpression suppressed the apoptosis of Ang II-induced HUVECs (), as displayed by the quantification (). To further confirm the effects of FGFR2 on cell apoptosis, the expression of apoptosis-related proteins was analyzed by western blot. It was discovered that the expression of Bax and cleaved PARP were upregulated and Bcl-2 expression was downregulated in Ang II-induced HUVECs, which were reversed by FGFR2 overexpression (). Caspase 3 is a prominent mediator of apoptosis. As expected, Ang II-enhanced caspase 3 activity in HUVECs was also depleted by FGFR2 up-regulation (). In summary, FGFR2 might protect against Ang II-stimulated HUVECs apoptosis.
Figure 3. Overexpression of FGFR2 inhibited Ang II-induced apoptosis in HUVECs. (A) the apoptosis of Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by Flow cytometry analysis and (B) the quantification. (C) the expression of apoptosis-related proteins in Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by western blot. (D) the caspase 3 activity in Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by caspase 3 activity assay kit. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group. ###P<.001 vs. Ang II + Oe-NC group.
Overexpression of FGFR2 alleviated Ang II-induced oxidative stress in HUVECs
As reported, oxidative stress is associated with the pathogenesis of hypertension. Here, the influences of FGFR2 on the oxidative stress in Ang II-stimulated HUVECs were estimated. Excess production of reactive oxygen species (ROS) has been defined as a contributor to oxidative stress. As displayed, Ang II elevated the activity of ROS in HUVECs, which was then diminished when FGFR2 was up-regulated. Additionally, MDA, CAT and GSH-Px are pivotal oxidative stress markers. Ang II challenging fortified the level of MDA while suppressed the activities of CAT and GSH-Px in HUVECs. Overexpression of FGFR2 could decrease the level of MDA and increase the activities of CAT and GSH-Px in Ang II-induced HUVECs (). To be concluded, FGFR2 up-regulation might ameliorate Ang II-triggered oxidative stress in HUVECs.
Figure 4. Overexpression of FGFR2 alleviated Ang II-induced oxidative stress in HUVECs. (A) the ROS level in Ang II-induced HUVECs transfected with Oe-FGFR2 was observed by DCFH-DA assay. Magnification, x400. (B) the oxidative stress levels in Ang II-induced HUVECs transfected with Oe-FGFR2 were detected by CAT assay kit, GSH-Px assay kit and MDA assay kit. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group. ###P<.001 vs. Ang II + Oe-NC group.
Overexpression of FGFR2 improved Ang II-induced endothelial dysfunction in HUVECs
Hypertension has been revealed to be associated with cardiovascular complications, possibly related to endothelial damage or dysfunction, or to abnormal angiogenesis. Subsequently, HUVECs migration and tube formation were respectively evaluated by wound healing and tube formation assays. It was noted that Ang II treatment attenuated the migratory capacity of HUVECs while FGFR2 overexpression potentiated the migration of Ang II-exposed HUVECs again (). Moreover, the tube formation ability of HUVECs was inhibited by Ang II challenging and FGFR2 overexpression improved the tube formation ability of Ang II-induced HUVECs (). In addition, the enzymatic production of NO by eNOS is critical in mediating endothelial function. NO level and p-eNOS/eNOS expression in Ang II-induced HUVECs were lower than that in HUVECs. FGFR2 overexpression increased NO level () and p-eNOS/eNOS expression () in Ang II-induced HUVECs. Accordingly, FGFR2 might mitigate Ang II-elicited endothelial dysfunction in HUVECs.
Figure 5. Overexpression of FGFR2 improved Ang II-induced endothelial dysfunction in HUVECs. (A) the migration of Ang II-induced HUVECs transfected with Oe-FGFR2 was analyzed by wound healing assay. Magnification, x100. (B) the tube formation ability of Ang II-induced HUVECs transfected with Oe-FGFR2 was analyzed by tube formation assay. Magnification, x400. (C) the NO level in Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by NO assay kit. (D) the expression of p-Enos and eNOS in Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by western blot. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group. ###P<.001 vs. Ang II + Oe-NC group.
Overexpression of FGFR2 regulates the Akt/Nrf2/antioxidant response element (ARE) signaling pathway in Ang II-induced HUVECs
More intriguingly, western blot analyzed the expression of Akt/Nrf2/ARE signaling-associated proteins and it was noticed that Ang II downregulated the expression of p-Akt/Akt, Nrf2, NQO-1 and HO-1 in HUVECs, which was not obviously affected by Oe-NC transfection. However, FGFR2 overexpression promoted the expression of p-Akt/Akt, Nrf2, NQO-1 and HO-1 in Ang II-induced HUVECs (), implying that FGFR2 activated the Akt/Nrf2/ARE signaling in Ang II-treated HUVECs.
Figure 6. Overexpression of FGFR2 regulates the Akt/Nrf2/ARE signaling pathway in Ang II-induced HUVECs. The expression of Akt/Nrf2/ARE signaling pathway related proteins in Ang II-induced HUVECs transfected with Oe-FGFR2 was detected by western blot. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group. ###P<.001 vs. Ang II + Oe-NC group.
Akt inhibitor MK-2206 reversed the protective effect of FGFR2 overexpression on Ang II-induced HUVECs
To validate that FGFR2 protected against Ang II-treated HUVECs injury through regulating Akt/Nrf2/ARE signaling, Akt inhibitor MK-2206 was adopted. As exhibited in , the experimental data from flow cytometry analysis manifested that FGFR2 overexpression inhibited the apoptosis of Ang II-treated HUVECs, which was then reversed by MK-2206, which could be obviously noticed in the quantification (). Also, FGFR2 elevation resulted in the decrease on the augmented Caspase 3 activity, Bax and cleaved PARP expression and the increase on the depleted Bcl-2 expression in HUVECs imposed by Ang II treatment. However, the impacts of FGFR2 on the Bcl-2, Bax, cleaved PARP expression () and Caspase 3 activity () were partially abolished by MK-2206. The levels of ROS () and MDA were decreased and levels of CAT and GSH-Px () were increased in Ang II-induced HUVECs transfected with Oe-FGFR2, which were reversed by MK-2206. FGFR2 overexpression promoted the migration () and tube formation ability () of Ang II-induced HUVECs, which was suppressed by MK-2206. FGFR2 up-regulation raised NO level and p-eNOS expression in Ang II-induced HUVECs and MK-2206 could downregulate NO level () and p-eNOS expression () in Ang II-induced HUVECs transfected with Oe-FGFR2. All in all, FGFR2 activated Akt/Nrf2/ARE signaling to ease Ang II-treated HUVECs injury.
Figure 7. Akt inhibitor MK-2206 reversed the protective effect of FGFR2 overexpression on apoptosis of Ang II-induced HUVECs. (A) the apoptosis of Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was detected by Flow cytometry analysis and (B) the quantification. (C) the expression of apoptosis-related proteins in Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was detected by western blot. (D) the caspase 3 activity in Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was detected by caspase 3 activity assay kit. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group. #P<.05, ##P<.01 and ###P<.001 vs. Ang II + Oe-FGFR2 group.
Figure 8. Akt inhibitor MK-2206 reversed the protective effect of FGFR2 overexpression on oxidative stress and endothelial dysfunction of Ang II-induced HUVECs. (A) the ROS level in Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was observed by DCFH-DA assay. Magnification, x400. (B) the oxidative stress levels in Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II were detected by CAT assay kit, GSH-Px assay kit and MDA assay kit. (C) the migration of Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was analyzed by wound healing assay. Magnification, x100. (D) the tube formation ability of Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was analyzed by tube formation assay. Magnification, x400. (E) the NO level in Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was detected by NO assay kit. (F) the expression of p-Enos and eNOS in Oe-FGFR2 transfected HUVECs treated by MK-2206 and Ang II was detected by western blot. Data from three independent replicates were presented as mean ± SD. ***P<.001 vs. Control group. ##P<.01 and ###P<.001 vs. Ang II + Oe-FGFR2 group.
Discussion
Hypertension is a common chronic disease. Surveys show that the incidence of hypertension in developed countries such as Europe and the United States is as high as 20%, with clinical characteristics of elevated blood pressure and cardiac and cerebrovascular disease (Citation13,Citation14). However, the molecular mechanisms underlying its pathogenesis are unclear. As an important component of blood circulation and the immune system, vascular endothelial cells are involved in a wide range of biological processes, including the regulation of blood pressure, angiogenesis, fibrinolysis and inflammation (Citation15,Citation16). A previous study has shown that vascular endothelial cell dysfunction plays an important role in the occurrence and development of hypertension (Citation17). Endothelial cells not only act as a physical barrier, but also secrete a variety of substances that affect contraction-relaxation function. Endothelial cell damage increases during hypertension (Citation18). Therefore, the study of the molecular mechanism of vascular endothelial cells in the occurrence and development of hypertension is helpful to improve the level of diagnosis and treatment of hypertension. In this study, Ang II-induced HUVECs were used to simulate the hypertension model in vitro, and it was found that the viability was reduced while oxidative stress levels and apoptosis was increased of HUVECs induced by Ang II, which indicated that the model was successfully established.
FGFRs is a protein family with 4 members and members of the FGFR family are tyrosine kinase receptors that play important roles in cell proliferation, differentiation, angiogenesis, bone development, and growth and development-related processes (Citation19,Citation20). A study has shown that FGFs/FGFRs signaling is closely related to cardiovascular diseases (Citation21). bFGF is a vasogenic multipotent growth factor that mainly acts on vascular smooth muscle cells to promote their proliferation and differentiation (Citation22), so it is particularly closely related to cardiovascular diseases. A previous study indicated that CD44 modulated the function of vascular endothelial cells by mediating FGFR2 activation and CD44 promoted plasma exosome proangiogenic function by enhancing FGFR2 signaling transduction (Citation23). Downregulation of adenosine deaminase acting on RNA 1 (ADAR1) promoted cell apoptosis and permeability of HUVECs by inhibiting FGFR2 (Citation24). FGFR2 inhibition suppressed the proliferation, migration, and angiogenesis of high glucose-induced human retinal microvascular endothelial cells (hRMECs) (Citation25). Promoting Ang II-induced HUVECs proliferation, inhibiting apoptosis and oxidative stress, and improving Ang II-induced HUVECs dysfunction could protect against Ang II-induced hypertension (Citation11). NO, a vital endogenous vasodilator, is produced in endothelial cells by eNOS, but its availability is impaired in various cardiovascular diseases (Citation26). In addition, previous studies have demonstrated that Ang II obviously suppressed NO synthesis in HUVECs by increasing the generation of ROS and sequentially inducing injury of endothelial cells (Citation27,Citation28). The present study indicated that FGFR2 overexpression improved the viability, migration and tube formation while suppressed the oxidative stress and apoptosis of Ang II-induced HUVECs.
FGFRs could activate the expression of Akt and ERK (Citation29). Activation of FGFR2-Akt-mammalian target of rapamycin (mTOR)-Nrf2 signaling pathway could protect against Ultra-violet induced injury in human retinal pigment epithelial cells (Citation30). We speculated that FGFR2 may regulate Akt/Nrf2 signaling pathway in Ang II-induced hypertension. Studies have found that the regulation of PI3K/Akt signaling pathway, one of the important signaling pathways for the prevention and control of hypertension diseases, can achieve the regulation of blood pressure in many aspects, such as repairing vascular endothelial function and inhibiting myocardial cell apoptosis (Citation31,Citation32). The therapeutical effect of electroacupuncture, dipeptide IF and exercise training on the hypertension was related to the activation of PI3K/Akt signaling pathway (Citation33,Citation34). Dysregulation of Nrf2 signaling pathway may lead to oxidative stress and endothelial cell dysfunction in hypertension. LopesRA et al. (Citation35) investigated the molecular mechanism of redox signaling in vascular arterioles and smooth muscle cells in stroke-prone spontaneously hypertensive rats, and determined that the downregulation of Nrf2 and its regulated antioxidant enzyme activity would reduce antioxidant capacity, increase oxidative stress, and lead to vascular dysfunction. Nrf-2, as an important regulator of endogenous antioxidant defense, mediates the level of HO-1 and other antioxidant enzymes, such as NQO1, CAT and GSH (Citation36,Citation37). Activation of Nrf2/HO-1 signaling pathway could improve vascular dysfunction and remodeling in hypertensive rats (Citation38). Here, we found that Akt/Nrf2/ARE signaling pathway was inhibited by Ang II in HUVECs and FGFR2 overexpression improved the viability, migration and tube formation while suppressed the oxidative stress and apoptosis of Ang II-induced HUVECs by activating the Akt/Nrf2/ARE signaling pathway. MK-2206, an Akt inhibitor, indeed reversed the impacts of FGFR2 overexpression on cell activity, migration, tube formation, oxidative stress and apoptosis of Ang II-induced HUVECs.
In conclusion, the expression of FGFR2 was decreased in Ang II-induced HUVECs. FGFR2 activated the Akt/Nrf2/ARE signaling pathway to improve Ang II-induced hypertension-related endothelial dysfunction.
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No potential conflict of interest was reported by the authors.
Additional information
Funding
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