Caspase-1 Mediates Hyperlipidemia-weakened Endothelial Progenitor Cell Vessel Repair

Section ?:IntroductionHyperlipidemia occurs due to the disorder of human lipid metabolism,which causes the blood lipid concentration to exceed the normal level.It is an important risk factor for the occurrence and development of many diseases,such as atherosclerosis,coronary atherosclerotic heart disease,myocardial infarction,cerebral infarction,and other diseases.It is characterized by lipid accumulation,inflammatory cell infiltration,and smooth muscle cell proliferation.In today's society,due to factors such as genetics,diet,nutrition,and drugs,the incidence of hyperlipidemia is increasing year by year,and there is a trend of the younger generation,which has become one of the most common diseases in humansMyocardial infarction(MI)is one of the significant complications of the occurrence and development of hyperlipidemia.Related studies have found that the risk of cardiovascular disease(CVD)in patients with hyperlipidemia is about twice that of people with normal cholesterol levels.After MI,ventricular remodeling will cause the heart chamber to expand,and the heart function declines.In recent years,studies have found that angiogenesis after MI plays a vital role in the recovery of heart function,and stem cell transplantation can reverse ventricular remodeling after MI by inducing angiogenesis and has a good effect in improving heart function and reducing pathological damage.Effect and clinical application prospectsUnder physiological conditions,the integrity of the endothelial cell monolayer is maintained by the replication of neighboring cells.However,in increased endothelial cell damage,the regeneration of endothelial cells is guided by endothelial progenitor cells(EPCs)Promoted into the blood vessel wall.EPCs are a group of hematopoietic stem cells that have the potential to differentiate into endothelial cells.Under stimulating factors such as ischemia,hypoxia,and hyperlipidemia,EPCs can be mobilized from the bone marrow,migrate to the injured area,and differentiate into mature endothelial cells and Paracrine a series of cell growth factors to promote endothelial repair and angiogenesis.Relevant clinical studies have shown that autologous transplantation of damaged EPCs can effectively improve the heart function and prognosis of MI patients,but the death rate of stem cells transplanted into the body is as high as 90%within one week.MI patients are mostly accompanied by hyperlipidemia,and the high-fat environment has a pro-apoptotic effect on EPCs,but the mechanism of high-fat-induced apoptosis of EPCs is not completely clear.Caspase-1 is a widely studied inflammatory-mediated Caspase that can cleave and activate inflammatory cytokines.Studies have found that Caspase-1 can activate cytokines such as IL-1? and IL-18,and IL-1? can affect the metabolism of triglycerides(TG)by interfering with the transmission and production of insulin signals,and can also directly inhibit lipids.Lipoprotein lipase(LPL)cycle.In animal experiments,it was also found that the clearance rate of TG in mice lacking Caspase-1 was increased.Therefore,in this experiment,we will explore whether we can increase the vascular repair effect of EPCs after myocardial infarction by reducing the activity of Caspase-1.Therefore,we put forward the following hypothesis:by inhibiting the expression of Caspase-1 from increasing the survival rate of EPCs,and ultimately promoting the regeneration of blood vessels after myocardial infarction,and improve heart function.In order to verify our hypothesis,in vivo experiments,we used high-fat feeding to construct a hyperlipidemia mouse model,and in vitro experiments used oxidized low-density lipoprotein(Ox-LDL)to intervene EPCs to clarify the effect of Caspase-1 on EPCs.At the same time,we constructed a mouse myocardial infarction model by ligating the anterior descending branch of the mouse and injected Caspase-1-/Caspase-1+EPCs into mice respectively to explore the role of EPCs in vascular repair after myocardial infarction.Section ?:High lipid induces endothelial progenitor cell apoptosis and elevated Caspase-1 expressionObject:To explore that high liquid can induce the enhancement of Caspase-1 activity and weaken the survival rate of EPCs.Method:1.The hyperlipidemia model of mice was constructed.Feed C57BL/6 wild type(WT)with high fat(HF)for six weeks.2.Extract EPCs:Anesthetize mice with pentobarbital,collect peripheral blood from the heart and wash the bone marrow cavity of both femurs and tibias of mice,obtain peripheral blood cells and bone marrow cells,and extract mouse mononuclear by density gradient centrifugation Cells(Mononuclear cells,MNC).3.Flow cytometry to detect bone marrow(BM)stem cell apoptosis:select EPCs cultured to the 7th day,and use different concentrations of Ox-LDL(0ug/ml,1ug/ml,10/ml,100ug/ml))Treat the cells for 24 hours,and detect the apoptosis level of BM stem cells by flow cytometry.4.Flow cytometry to detect EPCs:Flow cytometry analysis to detect Stem cell antigen-1(Sca-1+)and Vascular Endothelial Growth Factor Receptor 2(VEGFR2)/Flk-1 The cells that are all positive are EPCs;compare the ratio of BM-derived EPCs/BM stem cells,the mortality of EPCs(dead violet cells staining)and the cysteine protein hydrolase-1(Caspase-1)under high-fat or Ox-LDL stimulation-1)activity.Result:1.High fat can induce apoptosis of EPCs derived from mouse bone marrow,resulting in a significant decrease in their number(P<0.05),but has no significant effect on EPCs in peripheral blood(P>0.05);Caspase-in EPCs in HF-fed mice The activity of 1 is significantly increased.2.Ox-LDL can induce apoptosis of EPCs,and the treatment concentration of 100ug/ml is the best.In addition,the activity of Caspase-1 in EPCs increased after Ox-LDL stimulationConclustion:High lipid reduces the survival rate of EPCs by activating Caspase-1Section ?:The mechanism of endothelial progenitor cells promoting angiogenesis after myocardial infarction in miceObjective:To explore that Caspase-1-/-derived endothelial progenitor cells can improve the cardiac function of mice after myocardial infarction by reducing the apoptosis of myocardial cells and inducing blood vessel formationMethod:1.Construct a mouse model of acute myocardial infarction.Ligation of the left anterior descending coronary artery of the mouse to establish a mouse myocardial infarction model.Two weeks after the operation,blood was collected from the mice through the inner canthal vein,the mice were sacrificed,and the mouse hearts were collected.2.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)and Western bolt to identify the expression of Caspase-1.Cut mouse ear tissue,extract tissue DNA and tissue protein,qPCR and Western bolt to detect the mRNA and protein expression level of mouse Caspase-13.Magnetic beads sort mouse bone marrow-derived Sca-1+cells.8-10 weeks old wild type(WT)and Caspase-1 knockout mice were selected to obtain BMs of the tibia and fibula of the lower limbs of the mice.MNCs were separated from the BMs by density gradient centrifugation.Stain MNC with anti-Ly-6A/E(Sca-1)antibody,specifically label cells with superparamagnetic MACS micro-magnetic beads and place them in a sorting column a strong and stable magnetic field for sorting Sca-1 positive cells are detected.Finally,the purity of cell separation is tested by flow cytometry.4.Inject cells through the periorbital blood vessel.After pentobarbital anesthetized the mice to construct the myocardial infarction model,WT,and Caspase-1-Sca-1+cells derived from mouse bone marrow were injected through the periorbital artery,and the peripheral parts of the mice were taken 24h/48h/72h after the injection.Blood smears and flow cytometry were performed to detect the circulation of Sca-1+cells in the peripheral blood.At the same time,these mice were photographed in vitro to track the movement direction of Sea-1+cells.5.Echocardiography to detect the heart function of mice with myocardial infarction after Sca-1+cell therapy.M-mode echocardiography was performed on mice 14 days after myocardial infarction to detect left ventricular ejection fraction(LVEF)and left ventricular short-axis shortening rate(LVESD).6.After 14 days,blood was collected from the intracanthal vein,EPCs in the peripheral circulation of the mice were detected by flow cytometry,and the triglycerides(TG),total cholesterol(TC)and free of the mice were detected by a biochemical analyzer.Free fatty acids(FFA).7.Vessel regeneration after myocardial infarction in mice.The mice at the second weekend after myocardial infarction were sacrificed,and their hearts were taken,paraffin sections were made,and stained with IB4 and DAPI to observe the density of new blood vessels in the mice.Then,the paraffin sections of the mouse hearts were stained with TUNNEL to detect the mortality of mouse cardiomyocytes.Result:1.Connect the ECG to the mice during myocardial infarction surgery.The ECG indicates that the ST-segment elevation in lead ? indicates that the myocardial infarction model is successfully constructed.2.There is no Caspase-1 band detected by RT-PCR and Western blot,indicating that the Caspase-1 gene-deficient mice were successfully constructed.3.Bone marrow-derived Sca-1+cell therapy can simultaneously mobilize EPCs in mice,but cannot improve the levels of TC,TG and FFA in mice.4.Bone marrow-derived Sca-1+cell therapy can promote vascular regeneration after myocardial infarction in mice and improve heart function.The bone marrow-derived MNC is stained with anti-Ly-6A/E(Sca-1)antibody,and magnetic bead sorting can purify Sca-1+cells to about 85%.Two weeks after the injection of Sca-1+cells through the periorbital artery,the location of Sca-1+cells was tracked by X-ray in vitro,and it was found that most of the Sca-1+cells gathered in the heart cavity.Compared with mice injected with WT mouse Sca-1+cells,the LVEF and LVESD of the mice injected with Caspase-1-mouse Sca-1+cells were significantly improved,with statistical significance;and the latter mice The immunofluorescence staining of heart slices indicated that the capillary density and the survival rate of cardiomyocytes were significantly higher than the former(P<0.05).Conclusion:In the condition of myocardial infarction,exogenous injection of Caspase-1-/-derived Sca-1+cells can induce the mobilization of EPCs in mice,while reducing the mortality of EPCs,thus promoting the angiogenesis after myocardial infarction and improving cardiac function.