Study Of The Pathophysiological Mechanism By Which Apatinib Promotes Elevated Blood Pressure Through Inhibition Of Nitric Oxide Production By Vascular Endothelial Cells

Background With the development of oncologic cardiology,cardiovascular injury associated with oncologic therapy has received much attention.Studies have found that tumor patients using Vascular Endothelial Growth Factor Receptor 2(VEGFR-2)inhibitors have a higher incidence of hypertension,and apatinib(Apa),a VEGFR-2 inhibitor,is widely used in a variety of Apatinib(Apa),a VEGFR-2 inhibitor,is widely used in a variety of solid tumors,and treatment-induced hypertension has become one of the main reasons limiting its application.The mechanisms underlying the elevation of blood pressure in tumor patients due to Apa are not yet clear,so it is urgent to explore the pathophysiological mechanisms underlying the development of hypertension due to Apa and to find relevant protective drugs.Objective To investigate the role and molecular mechanism of nitric oxide(NO)in Apa-induced hypertension and the protective effect of NO donor L-arginine(L-Arg)on vascular function.Methods1.Establishment of cellular model Human Umbilical Vein Endothelial Cells(HUVEC)were randomly divided into Apa group(30 μM Apa treatment for 24 hours),Apa+L-Arg group(30 Apa treatment for 24 hours + 0.3 m M L-Arg for 30 minutes)and control group.control group,n=3 in each group.2.Establishment of animal models In this experiment,C57BL/6J mice were randomly divided into Apa low dose group(65 mg/kg/day),Apa medium dose group(110 mg/kg/day),Apa high dose group(155 mg/kg/day)and saline control group,with n=6 in each group.After the dose-dependent experiment,the best dose was selected: Apa medium dose group(110 mg/kg/day),and L-Arg(125 mg/kg/day)was given for intervention,n=6 for each group.3.Blood pressure monitoring in mice Diastolic pressure(DBP),systolic pressure(SBP)and mean arterial pressure(MAP)were monitored by a rat tail blood pressure meter.4.NO level measurement NO levels were measured in the supernatant of each HUVEC culture medium and in the serum of each group of mice by NO kit.5.Protein immunoblotting method The expression levels of HUVEC,aortic vascular phosphorylated nitric oxide synthase Ser1177 and its upstream pathway protein VEGFR-2 and p-Akt protein were investigated by protein immunoblotting.Apply protein immunoblotting to probe the expression levels of proteins of aortic vascular CD31.6.Histopathological analysis of aortic vessels Hematoxylin-Eosin Staining(H&E)staining and CD31 immunohistochemistry were applied to observe the pathological changes of aortic vessels in each group of mice.7.Statistical analysis All data of this experiment were expressed as mean ± standard error.Small sample t-test was used for comparison between two groups,and analysis of variance(ANOVA)was used for comparison between multiple groups.The data were imported into Graph Pad Prism 9.4.0 for plot analysis,and P<0.05 was statistically significant.Results1.Apa induces an increase in blood pressure in mice Apa induced a dose-dependent increase in blood pressure in mice,as evidenced by the increase in SBP,DBP and MAP,and the increase in blood pressure was reversed in the Apa medium-dose group with L-Arg.2.Apa can cause a decrease in NO production In in vivo experiments,serum NO levels were reduced in the Apa low-dose group,Apa mid-dose group and Apa high-dose group in a dose-dependent manner compared with the control group.In in vitro experiments,NO levels were reduced in the Apa group compared with the control group.3.Apa inhibits VEGFR/AKT/eNOS pathway In in vivo experiments,the aortic vascular Ser1177 and its upstream VEGFR-2 and p-Akt protein expression levels were significantly reduced in the Apa low dose group,Apa medium dose group and Apa high dose group compared to the control group.In in vitro experiments,the expression levels of Ser1177 and its upstream VEGFR-2 and p-Akt protein were significantly reduced in the Apa group compared with the control group.4.Apa leads to aortic vascular remodeling in mice Hematoxylin eosin(H&E)staining showed an increase in the ratio of intima-media thickness to lumen internal diameter(MT/LD)in mice with medium and high doses of Apa.CD31 immunohistochemical staining and immunoprotein blotting showed that the expression of CD31 was significantly lower in the aortic vessels of mice with low,medium and high doses of Apa compared with the control group.5.L-Arg reversed Apa-induced reduction in NO production,blood pressure elevation and vascular remodeling In in vivo experiments,L-Arg intervention reversed the increase in blood pressure and MT/LD in the Apa group mice on top of Apa application.Subsequent in vivo and in vitro experiments revealed that L-Arg improved the decrease in NO levels.Conclusion This study provides a new theoretical basis for the treatment of hypertension caused by VEGFR-2 inhibitors with L-Arg.Apa can disrupt endothelial function by inhibiting the VEGFR/AKT/e NOS signaling pathway,resulting in a decrease in the production of NO,a diastolic substance,which leads to impaired vascular diastolic function and ultimately hypertension and arterial remodeling,which can also aggravate vascular diastolic dysfunction and further aggravate hypertension.Arterial remodeling can also aggravate the diastolic dysfunction of blood vessels,further aggravating hypertension.L-Arg is expected to be a therapeutic agent for VEGFR-2inhibitor-induced hypertension because it reverses the decrease in NO production,increase in blood pressure and vascular remodeling caused by Apa,thus providing vascular protection.