The Mechanism And Intervention In Microvascular Dysfunction Of Coronary Slow Flow

Coronary microvascular dysfunction(CMD)was the main pathogenesis of nonobstructive coronary heart disease.Even with current optimal drug and interventional device strategies,the long-term prognosis of such patients was far from satisfaction,which lead to huge social and economic burden.Furthermore ischemic heart diseases derived from CMD had been at the top of the waiting list of clinical problems.Coronary slow flow phenomenon(CSFP)was chosen to be studied as one of the primary CMD subtypes in clinic,and such patients were enrolled from the inpatients from our hospital.In addition to the patients,CMD model was induced by lipid infusion in mice and challenged with palmitic acid(PA)in cultured cardiac microvascular endothelial cells(CMECs).Our study aimed to explore the potential pathological mechanisms about microvascular dysfunction of coronary slow flow,which would lead to deep understanding of CMD and provide with novel treatment strategies.Objective: Transcriptome sequencings and single-cell RNA sequencings were performed baesd on leukocytes derived from patients in two groups.Biochemical parameters of patients with coronary slow flow were compared with those in the control group in order to seek the hidden characteristics of CSFP.And in order to explore the mechanism of CMD,CMD mice model induced by lipid infusion and in vitro cultured cardiac microvascular endothelial cells were used.Methods:1.Whole transcriptome and single-cell sequencing of leukocytes of patients in Control(n=28)and CSFP group(n=28): Patients admitted to Department of Cardiology,Shanghai Changzheng Hospital from July 2017 to July 2019 and underwent coronary angiography were included in our study.Control and CSFP group patients were enrolled based on established inclusion and exclusion criteria from these included participants.Enrolled participants’ plasma and white blood cells were collected for the following investigations,including blood lipid analysis,whole transcriptome sequencing and single-cell RNA sequencing.Finally,we determined the plasma concentration of e NOS and s LOX-1 in the two groups of patients by ELISA.Concentration of free fatty acids(FFAs)was determined by microplate reader.2.CMD mice model induced by lipid infusion and the potential effects of nicorandil:30 C57 male mice(20±2g)of 6 weeks to 8 weeks were randomized into three groups,including control group,model and model+nico one.Mice in control group received intravenous infusion normal saline for 6 hours,while the model group and model+nico group were infused with a mixture of lipid(20%)and heparin,all of which were delivered via the jugular vein.Coronary flow reserve(CFR)was evaluated by ultrasound doppler.Glomerular filtration rate(GFR)was measured by transdermal GFR monitor.Cremaster muscle microcirculation(blood flow velocity,number of leukocytes adhesion and white blood cell rolling speed)was observed and recorded to the following analysis.The effects of lipid infusion on myocardial cells,renal podocyte,myocardial microvessels and gap junctions were evaluated by electron microscopy.Plasma samples were collected to determine the concentrations of FFAs,IFN-γ,IL-1β,IL-6,NO,SOD and TNF-α.Target genes of peripheral blood leukocytes were detected by quantitative PCR analysis,while ROS detection and flow cytometric analysis were investigated to uncover the potential mechanisms of CMD.Relative expressions of AMPK-KLF2-e NOS signal pathway were investigated of myocardial tissues derived from mice of different groups.3.Mechanism of CMD in mice and cultured cells: Peripheral blood neutrophils of CMD model induced by lipid infusion were used to explore neutrophil extracellular traps(NETs)production,and then the effects of NETs in leukocytes adhesion were studied with DNaseⅠ aiming to degrade NETs in circulation.Murine cardiac microvascular endothelial cells were cultured and challenged with different concentrations of PA for different time periods.Viability of CMECs and ROS production were evaluated by Cell Counting Kit-8(CCK-8)and ROS assay kit.The expression level of AMPK-KLF2-e NOS pathway signal in CMECs were measured by western blot.CMECs were preincubated with AICAR,KLF2 overexpression plasmids or nicorandil to study the potential roles of AMPK-KLF2-e NOS signal pathway.The viability and oxidative stress level were measured under different condition.And at last the protective effect of AICAR was examed in CMD mice models.Results:1.Whole transcriptome sequencing analysis showed that,compared with those of control group,there were 92 up-regulated transcripts and 129 down-regulated transcripts,which were involved with abnormal lipid metabolism and biological adhesion.In the CSFP group,the number of increased lnc RNAs was 738,while the decreased lnc RNAs was 472.In the CSFP group,a total of 117 circ RNAs were increased and 29 were decreased.Singlecell RNA sequencing analysis showed that genes related to the biological processes such as bioadhesion,antioxidant,immune response and metabolism were significantly changed in CSFP group.Compared with patients in the control group,patients in the CSFP group had lower plasma e NOS level and higher plasma FFAs and s LOX-1 concentration(P<0.05).Abnormal lipid metabolism,elevated plasma FFAs and endothelial dysfunction in patients with CSFP might be the typical characterstics of microvascular dysfunction of coronary slow flow and potential pathological mechanisms.2.Compared with those of the control group,the CFR and GFR of the mice in model group were significantly reduced.The number of adhered leukocytes,the expression levels of CD11 b and ROS concentrations were increased in the model group,but KLF2 m RNA levels and leukocytes rolling velocity decreased(P<0.05).Electron microscopy showed more swollen microvascular endothelial cells,ruptured gap junctions and damaged podocytes in the model group.Lipid infusion increased the concentrations of FFAs,TNF-αand IL-6.Nicorandil could significantly improve the CFR and GFR of CMD mice induced by intravenous infusion of lipid.It could also acclerate the leukocytes rolling speed,decrease ROS production and inflammatory factors secretion,reduce the adhered leukocytes.Relative expression levels of CD11 b were also reduced(P<0.05).Compared with those in the control group,the AMPK-KLF2-e NOS signal pathway was inhibited,while nicorandil could upregulate the expression level of e NOS in the heart and the concentration of NO in plasma(P<0.05).Intravenous infusion of lipid would induce CMD in mice and AMPK-KLF2-e NOS signal pathway inhibition played important role.Nicorandil might improve CMD by increasing NO concentration.3.NETs played an important role in the CMD induced by lipid,NETs eliminating could reduce the number of leukocytes adhered(P<0.05).PA had obvious concentration and timedependent effects on the viability reduction,oxidative stress level increase and AMPKKLF2-e NOS signal pathway inhibition in CMECs.Activating AMPK,KLF2 overexpression and activating e NOS all could alleviate the AMPK-KLF2-e NOS signal pathway inhibition induced by PA.Viability and oxidative stress level of CMECs were also improved(P<0.05).Activating AMPK improved CFR and leukocytes adhesion in model mice(P<0.05).Conclusion: Whole transcriptome sequencing analysis and single-cell RNA sequencing analysis showed that genes related to the biological processes such as lipid metabolism,antioxidant,and bioadhesion were significantly changed in CSFP group.Plasma FFAs and s LOX-1 concentration were increased and e NOS level decreased in CSFP group.Abnormal fatty acids metabolism,leukocytes adhesion and endothelial dysfunction might relate to the occurrence and development of microvascular dysfunction of coronary slow flow.FFAs could induce CMD in mice.The mechanism might be involved with abnormal inhibition of AMPK-KLF2-e NOS signal pathway.NO could improve CMD by reducing inflammatory cytokines,decreasing ROS and NETs production.