PD-L1 Regulates The Involvement Of Macrophage Metabolic Reprogramming In Neointima Formation After Vascular Injury

Object:Restenosis after percutaneous coronary intervention(PCI)can make a serious threat to patients’healthy after the procedure.The inflammatory response occurs rapidly after vascular injury and recruits large numbers of macrophages to the injury site.In response to external stimuli,macrophages can be reprogrammed into M1 and M2 macrophages through different metabolisms and participate in disease progression.Therefore,a clear understanding of macrophage metabolic reprogramming in post-PCI restenosis would be helpful for treatment and intervention.In our research,we initially explored the effects of altering levels of glucose metabolism in the injury microenvironment on macrophages and the modulating macrophage metabolic reprogramming on vascular smooth muscle cells(VSMCs)through ex vivo experiments,aiming to provide new therapeutic directions for the treatment of PCI restenosis.Methods:(1)Neointima formation in the sham-operated and operated groups was detected by HE staining.Glucose consumption,pyruvate production and lactate production in peripheral blood were detected by glucose oxidase method,pyruvate and lactate content test.Analysis of macrophage differences between sham-operated and operated groups by using GEO database.(2)Macrophages were cultured in a simulated metabolic alteration microenvironment.q RT-PCR was applied to detect changes in macrophage polarization-related IL-1β,TNF-α,IL-10 and TGF-βmRNA levels.Western Blot was used to detect macrophage PD-L1 protein expression.(3)Neointima formation in the DMSO and BMS-1 groups was detected by HE staining.Glucose oxidase assay,pyruvate and lactate content tests were used to detect the effect of BMS-1 on glucose metabolites in peripheral blood.(4)CCK8 to detect the effect of different BMS-1 administration concentrations on macrophage proliferation,flow cytometry to detect the effect of BMS-1 on macrophage PD-L1 expression and macrophage polarization.Glucose metabolites in macrophages and culture supernatant after the effect of BMS-1 by glucose oxidase assay,pyruvate content test and lactate content test.ATP content test was performed to detect the changes of ATP content in macrophages.q RT-PCR was performed to detect the mRNA expression of macrophage HK-2,PKM2 and PFK.Western Blot was performed to detect macrophage HK-2,PKM and PFK proteins.ELISA to detect the secretion of cytokines IL-1β,TNF-α,IL-10 and TGF-β1.(5)The proliferation ability of VSMCs was measured by CCK-8 assay and plate cloning assay;the migration ability of VSMCs was measured by cell scoring and Transwell;the expression of SMA,Vimentin and OPN in VSMCs was measured by Western Blot to further simulate the changes in the microenvironment after macrophage metabolism.The VSMCs were further cultured to mimic the changes of microenvironment after macrophage metabolism,and the proliferation ability of VSMCs was detected by plate cloning assay.The migration ability of VSMCs was detected by cell scoring and Transwell.Results:(1)HE staining results showed significant formation of new endothelium at 7d,14d,21d,28d and45d after vascular injury;glucose content in peripheral blood decreased(P<0.05),pyruvate and lactate content increased(P<0.05)from 1d to 45d after vascular injury,and macrophage infiltration in new endothelium increased.(2)In vitro mock glycolytic microenvironment cultured macrophages,along with increased lactate concentration,IL-1βand TNF-αmRNA decreased,IL-10,TGF-βand PD-L1 mRNA expression increased;lactate promoted PD-L1 protein level.(3)In vivo BMS-1 treatment was given locally at the injured vessels,and neoplastic endothelial proliferation was inhibited,promoting glycolysis levels in vivo;compared with the DMSO group,BMS-1 stimulation After BMS-1 stimulation,glucose levels decreased at 3d-45d(P<0.05),pyruvate levels increased at 8h,1d and 45d(P<0.001)and lactate levels increased at 0.5h-3d and 21d(P<0.01).(4)In vitro intervention of macrophage PD-L1 expression with BMS-1 showed a decrease in the percentage of PD-L1~+macrophages in flow(P<0.01)and promoted macrophage polarization towards M1 type(P<0.05);compared with DMSO control group,the intracellular and culture supernatant of macrophages in BMS-1 group showed decreased glucose content(P<0.05),increased pyruvate and lactate(P<0.05)and increased intracellular ATP content in macrophages(P<0.01);glycolysis-related enzymes HK-2,PKM2 and PFK mRNA and protein expression were increased(P<0.05);ELISA results showed increased expression of TNF-αand IL-1β(P<0.05)and decreased expression of IL-10 and TGF-β1(P<0.05).(5)Conditioned medium was prepared using BMS-1 group macrophage supernatant for VSMCs culture,and CCK8 and plate cloning assays was found to promote the proliferation ability of VSMCs(P<0.05),cell scoring and Tranwell results showed that VSMCs migration ability was inhibited(P<0.01);Western Blot assays revealed a decrease in the expression of contractile phenotype SMA and Vimentin proteins(P<0.01)and an increase in the expression of synthetic phenotype OPN protein(P<0.0001).The proliferation and migration of VSMCs were enhanced with increasing lactate concentration at 5 mM glucose and below 5 mM,while VSMCs proliferation and migration were inhibited at 10 and 20 mM lactate concentration.Conclusions:(1)Vascular injury will enhance glycolysis levels and promoted macrophage M2-type polarization and PD-L1 expression.(2)After vascular injury,inhibited PD-L1 can promote the glycolysis levels and inhibit neointima formation.(3)By affecting the biological function of VSMCs,inhibited PD-L1can promote macrophage glycolytic reprogramming and participate in neointima generation.