The Effect And Mechanism Of Human Urinary Kininogenase On Vascular Endothelial Injury Based On Pyk2/MCU Pathway

Cerebral infarction is a common and frequently encountered disease in the Department of Neurology.It has the characteristics of high incidence rate and high disability rate,which seriously affects the life quality of patients.Atherosclerosis is the most common cause of cerebral infarction.Endovascular therapy is a common approach in the current clinical treatment of cerebrovascular diseases,however,restenosis caused by endovascular therapy-induced injury influences the outcome of patients.The vascular endothelial cell injury is one of initiating factors in the development of atherosclerosis and the main cause of restenosis after endovascular treatment.Many factors can lead to endothelial cell injury,such as hyperglycemia,hypertension,inflammation,reactive oxygen species（ROS）and mechanical damage.The injury of endovascular therapy to vascular endothelium belongs to mechanical injury.With the increase of ROS around the injured vessels,the degree of endothelial cell injury becomes more serious.In the acute stage of cerebral infarction,endothelial cells began to proliferate,and then a large number of capillaries began to form.The formation of neovascularization can improve the blood supply to the brain,so as to promote the recovery of neurological function.Vascular endothelial growth factor（VEGF）is closely related to endothelial cell proliferation and angiogenesis.In conclusion,the occurrence and development of cerebral infarction is closely related to vascular endothelium.Up to now,the specific mechanism of vascular endothelial injury has not been fully understood.Therefore,in-depth study of the mechanism of vascular endothelial injury and how to protect vascular endothelial cells is of great significance for the prevention and treatment of cerebral infarction.Proline-rich tyrosine kinase 2（Pyk2）is a Ca²⁺-dependent tyrosine kinase.Previous studies have reported that Pyk2 is involved in the process of endothelial injury caused by factors including inflammation and diabetes.Furthermore,balloon injury causes activation of Pyk2,while blockade of Pyk2 can reduce intimal hyperplasia.Mitochondrial calcium uniport（MCU）,as the core component of the mitochondrial Ca²⁺uptake system,can regulate mitochondrial Ca²⁺homeostasis and apoptosis.The barrier effect of endothelium is one of the physiological functions of vascular endothelium.Previous studies have shown that inhibition of MCU can protect the endothelial barrier,indicating that there is a certain relationship between MCU and vascular endothelium.Our previous studies have shown that in cardiomyocytes,phosphorylated Pyk2 can activate MCU,which causes mitochondrial Ca²⁺overload,and initiates apoptosis signal transduction.However,the relationship between vascular endothelial cell injury and Pyk2/MCU pathway is not clear.Kinin plays an important role in vascular endothelial function.Human urinary kininogenase（HUK）is a tissue kallikrein,which can cleave inactive low-molecular-weight kininogen to release vasoactive kinin.Kinin binds to bradykinin receptors in vascular endothelium,triggering cascading biological effects,including inhibiting platelet adhesion and aggregation,promoting NO secretion,vasodilation and blood flow.In clinic,HUK is widely used in the treatment of cerebral infarction,and some studies have shown that HUK may be a potential therapeutic drug to prevent postoperative restenosis,however,the potential molecular mechanism between HUK and vascular endothelial injury has not been fully determined.The purpose of this study was to investigate whether the Pyk2/MCU pathway is involved in the process of vascular endothelial cell injury,and if HUK can protect vascular endothelial cells by inhibiting the Pyk2/MCU pathway,and if HUK can promote the recovery of neurological function in patients with cerebral infarction by regulating vascular endothelial function.Firstly,the carotid balloon injury model of male Sprague Dawley rats was established to explore whether the Pyk2/MCU pathway is involved in the process of vascular endothelial injury caused by balloon injury,and if HUK can protect vascular endothelial cells by inhibiting the Pyk2/MCU pathway.Because the vascular injury site caused by balloon injury is accompanied by the increase of ROS.Therefore,the oxidative injury model of vascular endothelial cells was established in vitro,in order to further explore whether the Pyk2/MCU pathway is involved in the process of vascular endothelial injury caused by oxidative damage and the protective mechanism of HUK on vascular endothelial cells.Then,the level of serum VEGF was detected in patients with acute cerebral infarction treated in the Department of Neurology of the Second Hospital of Hebei Medical University,in order to explore the effect of HUK on vascular endothelial function after cerebral infarction.The purpose of this study is to explore the effect and mechanism of HUK on vascular endothelial injury.It is divided into the following three parts,and the contents of each part are summarized as follows.Part One Human urinary kininogenase reduces vascular endothelial injury in rats after balloon injury by inhibiting Pyk2/MCU pathwayObjective:To investigate whether the Pyk2/MCU pathway is involv ed in the process of vascular endothelial injury caused by balloon injury,and if HUK can protect endothelial function via inhibiting the Pyk2/M CU pathway.Methods:The carotid balloon injury model was established.Rats w ere randomly divided into five groups:control group,sham group,ballo on injury group,HUK（0.00875PNA/kg/day）+balloon injury group,and HUK（0.0175 PNA/kg/day）+balloon injury group.At 7-and 14-days pos t-balloon injury,intimal hyperplasia was observed by hematoxylin and e osin staining（HE staining）.Cell ultrastructure was observed by transmis sion electron microscope（TEM）.The protein expressions of Pyk2,MCU and CD31 in the right common carotid artery of each group were exa mined by Western Blot on the 14th day after balloon injury.Results:1.On the 7th and 14th days after balloon injury,there was non-uniform thickening of the neointima and lumen stenosis of the common carotid artery in the injury group,the changes were more notable on the 14th day.HUK treatment markedly reduced neointimal formation and lumen stenosis on the14th day,but not on the 7th day,and there were no significant differences between the two HUK doses.2.Through TEM observation,this study found that the endothelial cell ultrastructure was normal in the control and sham groups,with a smooth cell surface and many pinocytotic vesicles.Compared with the control and the sham group,the ultrastructure of endothelial cells in the injury group changed significantly,the cell surface was broken and uneven,accompanied by the decrease or even disappearance of pinocytic vesicles,the expansion of endoplasmic reticulum and the swelling of mitochondria,and these changes were particularly obvious on the 14th day after balloon injury.However,HUK treatment markedly reduced balloon injury-induced cell ultrastructural damage on day 14,but not day 7.Furthermore,this effect was more notable in the high-dose group.3.The protein expression of Pyk2,MCU,and CD31 was no significant differences between the control and the sham group.However,the protein expression of Pyk2,MCU,and CD31 was significantly increased in the balloon injury group compared with the sham group.HUK treatment reduced the protein expression of Pyk2,MCU,and CD31 compared with the balloon injury group,but there were no significant differences between the two doses.Part Two Human urinary kininogenase reduces H₂O₂-induced vascular endothelial cell injury by inhibiting Pyk2/MCU pathwayObjective:To investigate whether the Pyk2/MCU pathway is involved in the process of vascular endothelial injury caused by oxidative damage,and the protective mechanism of HUK on vascular endothelial cells.Methods:The oxidative damage model of human umbilical vein endothelial cells（HUVECs）induced by hydrogen peroxide（H₂O₂）was established.The effect of HUK on cell viability after H₂O₂-induced HUVECs damage was observed through the MTS method.After 24 hours of synchronization,the cultured HUVECs were divided into four groups:control group,H₂O₂model group,HUK（0.25PNA/L）+H₂O₂group and HUK（0.5PNA/L）+H₂O₂group.The content of Ang II was detected by enzyme linked immunosorbent assay（ELISA）.The levels of ROS,mitochondrial membrane potential,intracellular free calcium and apoptotic cells were detected by flow cytometry.Cell ultrastructure was observed by transmission electron microscope（TEM）.The protein expressions of Pyk2,MCU and Caspase-3 was observed by Western Blot;Real time reverse transcription quantitative polymerase chain reaction（RT-q PCR）was used to detect the m RNA expression of Pyk2 and MCU.In addition,the expression of Pyk2 in HUVECs was inhibited by short hairpin RNA（sh RNA）technology,and Pyk2low expression cells were obtained.The cells were treated with H₂O₂and HUK again,and the cell viability was observed by MTS method.The level of intracellular free calcium was observed by flow cytometry.The protein expressions of Pyk2,MCU and Caspase-3 were observed by Western Blot.Results:1.HUK alleviates the decrease of HUVECs activity caused by H₂O₂partly through B2R,and inhibits the excessive production of AngⅡand ROS induced by H₂O₂,so as to protect vascular endothelial cells.2.HUK inhibits H₂O₂-induced cell apoptosis and intracellular free Ca²⁺overload,reduces mitochondrial damage and protects the cell ultrastructure in HUVECs.3.HUK inhibits the activation of Pyk2/MCU pathway in HUVECs induced by H₂O_2。4.Pyk2 low expression inhibits H₂O₂-induced cell death,intracellular free Ca²⁺overload,and the protein expression up-regulation of MCU in HUVECs.Part Three The effect of human urinary kininogenase on serum VEGF level in patients with acute cerebral infarctionObjective:To observe the effect of HUK on serum VEGF level in patients with acute cerebral infarction,and to explore the effect of HUK on vascular endothelial function after cerebral infarction.Methods:42 patients with acute cerebral infarction were enrolled in this study who hospitalized in the Department of Neurology,Second Hospital of Hebei Medical University from July 2017 to December 2018.And all patients were randomly divided into two groups.The experimental group was given HUK and standard treatment based on guide,while the control group was only given standard treatment based on guide,and both groups were treated for14±5 days.The clinical information of patients were collected,the neurological function of patients before and after 14±5 days treatment were evaluate,and the levels of VEGF in serum of patients were detected by ELISA.Results:1.There was no significant difference in clinical baseline data between the experimental group and the control group（P>0.05）.2.In the experimental group,there were significant differences in NIHSS score and BI index between 14±5 days after treatment and before treatment（P<0.05）.In the control group,the NIHSS score was significant difference between 14±5 days after treatment and before treatment（P<0.05）,but the BI index had no significant difference between 14±5 days after treatment and before treatment（P>0.05）.There was significant difference in NIHSS score difference between the experimental group and the control group after 14±5 days treatment（P<0.05）.3.There was no significant difference in the level of serum VEGF between the two groups before treatment（P>0.05）.The level of serum VEGF in the experimental group was significantly different between 14±5days after treatment and before treatment（P<0.05）,while the level of serum VEGF in the control group had no significant difference between 14±5 days after treatment and before treatment（P>0.05）.Conclusions:1.Pyk2/MCU pathway is involved in the process of endothelial injury of rat common carotid artery induced by balloon injury.HUK can reduce intimal hyperplasia caused by balloon injury and prevent restenosis after endovascular treatment by inhibiting Pyk2/MCU pathway.2.Pyk2/MCU pathway is activated in H₂O₂-induced vascular endothelial cell injury.Pyk2 low expression can reduce the injury of vascular endothelial cells induced by H₂O₂.HUK reduces H₂O₂-induced apoptosis and mitochondrial damage,protects vascular endothelial cells by inhibiting Pyk2/MCU pathway.3.HUK can improve vascular endothelial function and promote the recovery of neurological function by regulating the level of serum VEGF in patients with acute cerebral infarction.