The Mechanism And Intervention Of Tyrosine Kinase Inhibitors On Blood Pressure And Cardiac Injury Through RhoA/ROCK Pathway

Background and objective:Hypertension is one of the most common cause of adverse cardiovascular effects of targeted antineoplastic drugs.Tyrosine kinase inhibitors(TKIs)targeting vascular endothelial growth factor(VEGF)can achieve anti-tumor effects by inhibiting the phosphorylation level of tyrosine kinase,thereby inhibiting DNA repair of tumor cells,blocking G1 phase cell division,and inhibiting angiogenesis.Apatinib,a representative TKI,is used as a third-line or above treatment for advanced gastric cancer or gastroesophageal junction adenocarcinoma.However,the prevention and treatment is different from essential hypertension,that the incidence of hypertension is high,and the pathogenesis needs to be further studied.Therefore,it is of great value to study the mechanism of hypertension induced by apatinib in the treatment of cancer patients,to find drugs that can reduce blood pressure without affecting anti-tumor therapy,and to conduct mechanism and intervention experimental research.ROCK is mainly involved in the regulation of vasoconstriction.Y27632 is a selective smallmolecule inhibitor of ROCK1 and ROCK2 that inhibits both rat and human tumor cell erosion and metastasis.Therefore,Y27632 may counteract the blood pressure elevation effect of VEGF inhibitors and promote the anti-tumor effect of apatinib.Our team has previously found that apatinib can significantly increase blood pressure in WKY rats through the Rho A/ROCK signaling pathway,and previous studies have shown that sunitinib can aggravate the activation of the Rho A/ROCK signaling pathway induced by high-salt diet(HSD).Therefore,we hypothesized that apatinib could increase blood pressure and salt sensitivity in a real tumor model(gastric cancer model mice)through the Rho A/ROCK signaling pathway.The aim of this study is to investigate whether Rho A/ROCK signaling pathway is involved in the mechanism of apatinib-induced hypertension and whether it is one of the mechanisms of vascular and cardiac remodeling by simulating the tumor environment in vivo.At the same time,to explore whether ROCK pathway inhibitor Y27632 can prevent and reverse apatinib treatment of gastric cancer induced blood pressure elevation,vascular and cardiac remodeling,and to provide a theoretical basis for the prevention and treatment of this type of hypertension.Methods:A total of 56 female SPF Balb/c nude mice(20±5g)aged 4-6 weeks were randomly divided into normal(NR)group(n=8)and tumor-bearing(TB)group(n=48).Human gastric cancer cell line MKN-45 was used to establish tumor models in tumor-bearing mice.After successful modeling,the mice were randomly divided into 6 groups and were received drug intervention for 7 weeks:(1)Control group:gavage of 2ml normal saline;(2)high-salt diet(customized 8% high-salt diet);(3)high-salt diet(customized 8% high-salt diet)+Y-27632(10mg/kg?d)intraperitoneal injection;(4)High-salt diet(customized 8% high-salt diet)+ apatinib(50mg/kg?d)dissolved in 2ml normal saline by gavage;(5)Apatinib(50mg/kg?d)was dissolved in2 ml normal saline by gavage;(6)Apatinib(50 mg/kg)was dissolved in 2ml normal saline by gavage,and Y27632(10mg/kg?d)was injected intraperitoneally.The levels of systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean arterial pressure(MBP)were monitored at fixed time points every week.The cardiac function of mice in each group before and after drug intervention was evaluated by small animal ultrasound.Real-time fluorescent quantitative PCR(q RTPCR)and Western blot(WB)were used to detect the Rho A/ROCK signaling pathway,endothelial nitric oxide synthase(e NOS)and endothelin-1(ET-1)protein expression and corresponding m RNA levels,as well as smooth muscle myosin light chain(MLC)and myosin phosphatase targeting subunit 1(MYPT-1)and phosphorylated MLC and MYPT-1 protein levels.H&E staining was used to observe the structural changes of aorta and heart tissue,and Masson staining was used to observe the degree of fibrosis in aorta and heart tissue.The expression levels of collagen Iα1(COL1α1),collagen IIIα1(COL3α1)and smooth muscle actin(α-SMA)in aortic media and heart tissue were detected by immunohistochemistry,and the differences in myocardial fibrosis and vascular stiffness were compared.Immunohistochemical staining of platelet endothelial cell adhesion molecule(CD31)and e NOS was performed to observe the effect of apatinib on small vessel density in superior mesenteric arteries of mice.SPSS 23.0 software was used for statistical analysis,Image J software was used for semi-quantitative analysis of protein bands,and Graph Pad Prism8.0 was used for drawing.Results:1.Evidence for successful establishment of a mouse model of gastric cancer: The tumor volume of each group of gastric cancer model mice was more than 100mm3.The tumor tissues of each group of gastric cancer model mice were removed and H&E staining showed tumor cell-like changes,indicating that the gastric cancer mouse model was successfully established.2.Changes of tumor-volumein each group before death: The tumor volume of the apatinib group was significantly smaller than that of the control group(p<0.001).There was no significant difference in tumor-volum eafter combined Y27632intervention(p>0.05);The tumor volume of high-salt group increased most significantly,while the tumor volume of combined Y27632 intervention decreased significantly(p<0.001).3.Differences in body weight of mice in each group before sacrifice: Compared with a baseline body weight,the body weight increased significantly in the NR group and decreased significantly in the control group.The most significant decrease was observed in the high-salt group.The body weight increased significantly in the highsalt group after the intervention of apatiniband Y27632(p<0.001);However,the body weight of mice in the apatinib group was higher than that in the control group(p<0.001),but there was no significant difference in body weight after combined Y27632 intervention(p>0.05).4.Blood pressure trends of mice in each group: There was no significant difference in blood pressure(p>0.05).One week after the gastric cancer model was established,blood pressure showed a temporary increase and then returned to the baseline level at the third week.At 7-8 weeks,the blood pressure level reaches a plateau.Blood pressure in each group was as follows: NR group(118.00/68.00±1.03/2.00 mm Hg),and control group(104.00/58.00± 1.75/2.62 mm Hg),Blood pressure increased significantly in apatinib group(158.83/101.83 ± 2.71/1.54)and high-salt diet group(167.33/105.33 ± 2.11/5.78 mm Hg).In the high-salt group,the blood pressure(188.17/133.67 ± 2.07/1.17 mm Hg)showed the most significant increase after concurrent administration of apatinib,illustrating an additive effect(p<0.001),while,after Y27632 intervention,the blood pressure of high-salt diet group(101.00/57.00 ± 1.67/1.84 mm Hg)and apatinib group(98.17/66.17 ± 1.25/3.51 mm Hg)decreased significantly(p<0.001).5.The protein and m RNA levels of Rho A,ROCK1,ROCK2,P-MLC and PMYPT were significantly increased in the apatinib group and the high-salt diet group,while the protein levels of Rho A,ROCK1,ROCK2,p-MLC and p-MYPT were decreased in the apatinib group and the high-salt diet group after Y27632 intervention(p<0.001),which was more significant in the high-salt group when combined with apatinib(p<0.001).6.Changes of vascular endothelial relaxation-related factors in each group: The protein and m RNA levels of ET-1 and e NOS were significantly increased in the apatinib group and high-salt diet group,and the combination of Y27632 reduced the expression levels of ET-1 and Enos in the apatinib group and high-salt diet group(p<0.001),which was more significant in the high-salt group when combined with apatinib(p<0.001).7.Histological changes of aorta in each group of mice: In the high-salt group,the intervention of apatinib could make this phenomenon more obvious.In the high-salt group,the intervention of apatinib combined with Y27632 increased the lμmol/len area and reduce the blue-stained fibrotic area(p<0.001);Immunohistochemistry and q RT-PCR showed that the positive signals and m RNA levels of COL1 and COLIII in the aorta of mice in the apatinib group and high-salt diet group increased,and the positive signals and m RNA levels of COL1 and COLIII in the high-salt diet group increased more significantly after the intervention of apatinib.However,the levels of positive signals and and m RNAs were decreased after Y27632 intervention(p<0.001).8.Changes of small vessel density in each group: immunohistochemistry and q RT-PCR showed that the expression levels of e NOS and CD31 positive signals and m RNAs in the superior mesenteric arteries of mice were decreased after apatinib intervention,and were increased after Y27632 intervention(p<0.001).9.Cardiac remodeling changes of mice in each group: Echocardiography showed that left ventricular diastolic function indexes including left ventricular posterior wall thickness(LVPW),left ventricular end-diastolic diameter(LVIDd),left ventricular end-systolic diameter(LVIDs),heart weight(HM)and left ventricular mass(LVM)increased in the apatinib group and the high-salt diet group,but decreased after the intervention with Y27632(p<0.001);The increase was most significant after apatinib intervention in the high-salt diet group,showing an additive effect(p<0.001).Compared with the above indicators,the left ventricular ejection fraction(EF)and left ventricular fractional shortening(FS)showed an opposite trend in each group of mice(p<0.001).H&E and Masson staining of the heart showed that the apatinib and highsalt diet groups had disorganized the arrangement of cells,damaged the muscle fibers,widened the interstitial space,and increased the blue-stained area of cardiac fibrosis,which were most significant in the high-salt diet + apatinib group.After Y27632 intervention,the blue-stained area of myocardial fibrosis in the apatinib group and the high-salt diet group decreased(p<0.001).The results of immunohistochemistry and q RT-PCR showed that the positive signal of α-SMA and the m RNAs expression levels of COL1 and COLIII related factors in heart tissues of mice in the apatinib group and high-salt diet group were increased,while the positive signal and m RNAs expression levels of COL1 and COLIII related factors were decreased after Y27632 intervention(p<0.001).In the high-salt group,the levels of positive signals and m RNAs increased significantly after the intervention of apatinib,showing an additive effect(p<0.001).Conclusions:1.In the mouse model of gastric cancer,apatinib has a good therapeutic effect on tumors,and its combination with Y27632 does not affect the anti-tumor effect of apatinib.2.Apatinib can increase the blood pressure of gastric cancer mice by activating the Rho A/ROCK signaling pathway,and combined with the addition of Y27632 intervention can significantly reduce the apatinib-induced high blood pressure increase.3.Y27632 can reverse aortic vascular remodeling,superior mesenteric artery vascular injury and small vessel density sparse in a mouse model of gastric cancer induced by apatinib.4.Y27632 can improve left ventricular hypertrophy and fibrosis in a mouse model of gastric cancer induced by apatinib.5.Apatinib can increase the salt sensitivity of gastric cancer mouse model through the Rho A/ROCK signaling pathway,and therefore aggravate the development of hypertension,showing an additive effect.6.Y27632 may also have a certain therapeutic value for salt-sensitive hypertension induced by apatinib.Background and Objective:In the first part of the study,apatinib increased blood pressure in gastric cancer mice by inducing vascular remodeling and thinning of small vessel density through the Rho A/ROCK signaling pathway.The abnormal proliferation and invasion of vascular smooth muscle cells(VSMCs)is one of the main causes of hypertension and vascular remodeling.Therefore,activation of Rho A/ROCK signaling pathway may be one of the potential mechanisms of apatinib induced dysfunction in mouse aortic vascular smooth muscle cells(MOVAS).The leukocyte-related factor Rho guanosine transfer factor 12(ARHGEF12,LARG)is a key gene in the Rho A/ROCK signaling pathway.LARG is mainly expressed in blood vessels and is responsible for the activation of Rho A in response to mechanical strain.Because LARG can regulate part of VSMCs signaling and therefore plays a key role in vasoconstrictor function,the absence of LARG gene,reduces the abnormal increase in blood pressure but does not alter normal blood pressure regulation.Therefore,LARG-mediated signaling could be a novel target for antihypertensive therapy.VSMCs can switch to an undifferentiated synthetic phenotype under external stimulation,and further play the role of proliferation,migration and synthesis of extracellular matrix.This change will lead to abnormal proliferation of VSMCs.Proliferation and mitophagy play an important role in maintaining the normal function and structure of blood vessels.When the balance between proliferation and mitophagy is broken,the excessive activation of mitophagy will lead to energy damage and increased reactive oxygen species,which will further cause abnormal proliferation of VSMCs and induce hypertension.Therefore,we hypothesized that ROCK inhibitor Y27632 and LARG gene knockdown could improve the abnormal proliferation and invasion of MOVAS cells induced by apatinib,and LARG gene knockdown may also have potential therapeutic value for apatinib-induced hypertension.In this study,we investigated whether Y27632 combined with apatinib could alleviate apatinib induced MOVAS cell injury in vitro.We also observed whether the abnormal proliferation and invasion of MOVAS cells induced by apatinib can be improved by knocking down LARG gene.Methods:MOVAS cells were treated with different concentration gradients of apatinib(0,2.5,5,10)μmol/l for 48 hours,and the effects of apatinib on the proliferation,apoptosis and invasion of MOVAS cells were observed by cell counting kit-8(CCK8),flow cytometry,cell scratch test and immunofluorescence.At the same time,whether apatinib could affect the expression of vascular smooth muscle cell function related factors e NOS,ET-1 and α-SMA protein levels in MOVAS cells were detected.The protein and m RNA expression levels of Rho A/ROCK signaling pathway,e NOS,ET-1and α-SMA were detected by q RT-PCR and WB.Y27632(10μmol/l)was used to observe whether it could improve apatinib-induced MOVAS cell dysfunction.Lentivirus transfection was used to construct LARG gene knockdown cell lines,and different groups were set up: control group,apatinib group(10μmol/l),sh RNALARG gene knockdown group,and sh RNA-LARG gene knockdown + apatinib(10μmol/l)group.q RT-PCR and WB were used to detect the protein expression and corresponding m RNA levels of Rho A/ROCK pathway.Immunofluorescence was used to detect the effect of LARG gene on e NOS and ET-1 system in apatinib induced MOVAS cells.Finally,flow cytometry,immunofluorescence and mitochondrial membrane potential were used to observe the effect of LARG knockdown on MOVAS and whether it could reverse the abnormal proliferation,cycle changes and mitochondrial membrane potential changes of MOVAS caused by apatinib.SPSS23.0 software was used for statistical analysis,Image J software was used for semiquantitative analysis of protein bands,and Graph Pad Prism8.0 was used for drawing.Results:1.Changes of Rho A/ROCK signaling pathway: Different concentrations of apatinib could increase the expression of Rho A and ROCK2 m RNAs and proteins(p<0.001).The expression of ROCK1 m RNA and protein increased when the concentration reached 10μmol/l(p<0.001);Y27632 inhibited apatinib(10μmol/l)induced upregulation of Rho A and ROCK2 m RNAs and proteins(p< 0.001),but could not reduce the increase of ROCK1 m RNA and protein expression levels.2.CCK8,cell scratch test and flow cytometry results: With the increase of apatinib concentration,the proliferation rate of MOVAS increased gradually,and the area under the scratch decreased gradually(p< 0.001),while the apoptosis rate of MOVAS increased at only 10μmol/l(p< 0.001).After combined treatment with Y27632,the proliferation rate of MOVAS was inhibited under different concentration gradients,and the area under scratch and apoptosis rate of MOVAS were increased(p< 0.001).3.Immunofluorescence results showed the following: With the increase of apatinib concentration,the positive signal expression of α-SMA and e NOS gradually decreased,and the expression of ET-1 and i NOS gradually increased(p< P < 0.05).0.001);Combined with Y27632 intervention,the expression of α-SMA and e NOS gradually increased,and the expression of ET-1 and i NOS gradually decreased(p<0.001).4.Changes of Rho A/ROCK signaling pathway after LARG knockdown: WB and q RT-PCR results showed that sh RNA-LARG knockdown significantly reduced the m RNA and protein expression levels of Rho A,ROCK1,and ROCK2,as well as the protein levels of p-MLC and p-MYPT(p< 0.001).5.Cell cycle changes of MOVAS after LARG gene knockdown: Knockdown of sh RNA-LARG significantly reduced apatinib-induced increase in the proportion of MOVAS cells in S% phase +G2/M% phase(p< 0.001).6.Effects of LARG gene knockdown on apoptosis of MOVAS cells: Apatinib reduced the apoptosis rate of MOVAS cells,the expression level of caspase-3 protein,and increase the ratio of BAX/BCL2(p< 0.001);Knockdown of sh RNA-LARG significantly increased the apoptosis rate of MOVAS cells,the expression level of caspase-3 protein,and decreased the ratio of BAX/BCL2(p< 0.001).7.Effects of LARG gene knockdown on mitophagy in MOVAS cells: The protein levels and of beclin1 and LC3 II in sh RNA-LARG knockdown group were lower than those in control group(p< 0.001).LARG knockdown also improved beclin1,LC3 II protein levels and in MOVAS cells induced by Apatinib(p<0.001).8.Changes of ROS in MOVAS cells after LARG gene knockdown: Knockdown of sh RNA-LARG decreased ROS production in MOVAS cells and significantly improved the apatinib-induced ROS increase(p< 0.001).9.Effects of LARG gene knockdown on functional factors of MOVAS cells: The results of q RT-PCR and immunofluorescence showed that sh RNA-LARG gene knockdown increased the expression of α-SMA and e NOS positive signals in MOVAS cells,and decreased the expression levels of ET-1 and i NOS(p< 0.001);Knockdown of LARG also improved the apatinib-induced e NOS/i NOS imbalance(p< 0.001)Conclusions:1.Apatinib can enhance the proliferation rate,cell migration and anti-apoptosis ability of MOVAS through the Rho A/ROCK signaling pathway,leading to MOVAS cell dysfunction.Y27632 can improve apatinib-induced MOVAS cell dysfunction by inhibiting the activation of this signaling pathway.2.LRAG knockdown can not only shorten the growth cycle of MOVAS,increase the apoptosis of MOVAS and reduce the migration ability of MOVAS cells,but also improve the cycle prolongation,reduce the degree of apoptosis and abnormal increase of migration ability of MOVAS cells caused by apatinib.Therefore,LRAG knockdown can improve the abnormal proliferation of MOVAS cells induced by apatinib.3.Apatinib can increase the expression of autophagy-related proteins and change mitochondrial membrane potential causing cause mitophagy in MOVAS cells,while LRAG gene knockdown can alleviate apatinib induced mitophagy and further improve the damage of MOVAS cells.4.Knockdown of LRAG can reduce the increase of ROS in MOVAS cells induced by apatinib.Therefore,knockdown of LRAG may improve the oxidative stress response induced by apatinib.5.LRAG knockdown can improve the imbalance of i NOS/e NOS ratio and abnormal expression of ET-1 and α-SMA in MOVAS cells caused by apatinib.Therefore,knockdown of LRAG improves the abnormal contractile function and cell dysfunction of MOVAS cells induced by apatinib.6.By exploring the damage mechanism of apatinib on MOVAS cells found that the main mechanisms of apatinib induced MOVAS cell proliferation included decreased apoptosis rate,increased proliferation ability,increased migration ability,shortened cell proliferation cycle,triggered mitautophagy,increased expression of reactive oxygen species,cell phenotype switching and cell dysfunction.Knockdown of LRAG and administration of Y27632 could improve the above conditions.This study provides a therapeutic basis and direction for the treatment of cancer-induced hypertension with TKIs.Cardiovascular diseases and tumors are the two main causes of increased mortality worldwide.Nμmol/lerous studies have proved that there is a close relationship between them.However,with the advancement of tumor targeted therapy technology,the survival of cancer patients continues to be prolonged,and cardiovascular adverse effects have gradually become one of the important causes of death in cancer patients.The occurrence of this phenomenon is due to the cardiovascular toxicity of anti-tumor therapy,which leads to the occurrence and development of cardiovascular adverse effects.On the other hand,due to the extension of life expectancy,the proportion of elderly patients with cancer and cardiovascular diseases increases.tumor growth and survival depend on adequate blood supply from new blood vessels.As solid tumors grow,the central core becomes hypoxic,a process that allows the tumor tissue to release angiogenic growth factors and stimulate new blood vessel formation.These new blood vessels provide nutrients and water for tumor growth and spread tumor cells to distant sites.Anti-angiogenic drugs can specifically and targeted block tumor blood vessels by eliminating the blood vessels necessary for solid tumor growth and metastasis,so as to achieve the purpose of treating tumors.Vascular endothelial growth factor(VEGF)expressed by tumor cells is the most important proangiogenic factor,which can induce neovascularization in vivo to maintain tumor growth.By blocking the binding of VEGF to endothelial cell surface receptors,the formation of new blood vessels is inhibited.Therefore,inhibition of VEGF signaling has emerged as a major therapeutic target in cancer therapy.VEGF signaling pathway inhibitors include monoclonal antibodies against VEGFA,vascular endothelial growth factor inhibitors(VEGF trap),monoclonal antibodies against VEGF receptors,and small molecule tyrosine kinase inhibitors(TKIs).TKIs are effective signaling cascade inhibitors that inhibit tumor blood vessel growth by inhibiting vascular endothelial growth factor receptor(VEGFR).VEGFRTKIs have become the main treatment for many solid malignant tumors.However,TKIs can induce vascular endothelial damage,hypertension and myocardial injury by targeting VEGFR,platelet-derived growth factor receptor(PDGFR)and stem cell factor receptor(SCFR).It can also damage mitochondria and affect myocardial energy metabolism through the "off-target effect",eventually leading to cardiovascular complications.Therefore,the incidence of cardiovascular toxicity related to VEGFR-TKIs is high,which can cause the occurrence and development of cardiovascular complications such as hypertension,left ventricular systolic dysfunction/heart failure,and atherosclerosis.Cardiovascular toxicity and other side effects of anti-tumor therapy have become one of the main reasons limiting the development of anti-tumor therapy.Therefore,the goal of TKIs therapy is to maximize the anti-tumor effect and reduce the treatment-related cardiovascular events.The search for new specific therapeutic drugs is very important for the smooth application of anti-tumor therapy,the prevention of related cardiovascular events and the improvement of patient prognosis.