Effect And Mechanism Of PD-1 On Regulating Blood Profusion And Angiogenesis In Mice After Hindlimb Ischemia

ObjectiveAngiogenesis is an important process in both physiological and pathophysiological conditions.Abnormal angiogenesis contributes to many pathophysiological processes,such as stroke,myocardial infarction,limb ischemic and atherosclerosis.While the primary mechanism of ischemia-induced angiogenesis involves the production of HIF-1?protein,and the consequent increase of expression of VEGF.They induced the endothelial cells proliferation,migration and tube formation.Ischemia-induced angiogenesis is also modulated by the inflammatory response associated with the ischemia-induced tissue injury.Programmed cell death protein 1 is a protein on the cell membrane that plays an important role in immune tolerance through inhibiting both the initial induction and the maintenance of effective memory T cell tolerance and the formation and survival of Tregs.PD-1 is an immune checkpoint and exerts an important role in regulating autoimmunity.Inhibition of the PD-1 signaling pathway is one of the most effective approaches in treating cancers but anti-PD1 immunotherapy also results in various cardiovascular toxicities in some cancer patients.However,the role of PD-1 in hindlimb ischemia-induced inflammation and angiogenesis is unknown.Here we investigated the role and the underlying mechanism of PD-1 in hindlimb ischemia-induced inflammation and angiogenesis in mice.MethodsFemale wild type and PD-1 KO mice?Jackson Laboratory?of C57 background were used for hindlimb ischemia surgery.Laser-Doppler perfusion imaging were used to test the blood profusion before and after hindlimb ischemia surgery.Mouse exercise capacity were tested by treadmill.Flow cytometry,immunofluorescence staining,H&E staining,Masson staining,Real time PCR and ELISA were used for analysis.The proliferation,migration,tube formation and apoptosis of HUVEC were analysis after expose to IFN-?.Results1.PD-1^-/-impaired blood perfusion,muscle angiogenesis,exercise capacity,exacerbated mouse muscle dystrophy and fibrosis in mice after hindlimb ischemia.As compared with WT mice,blood perfusion in PD-1 KO mice were similar before hindlimb ischemia.However,the hindlimb blood perfusions were recovered significantly less in PD-1 KO mice 14 and 21 days after hindlimb ischemia,suggesting impaired angiogenesis capacity in PD-1 KO mice as compared with the corresponding WT mice.Since hindlimb blood perfusions in PD-1-/-mice were similar to WT mice up to 7 days after femoral artery ligation but were significantly impaired in PD-1^-/-mice as compared to WT mice 14 and 21 days after femoral artery ligation,to understand whether these changes were resulted from the differences in vessel regeneration,we further determined capillary density in PD-1-/-and WT mice 7and 14 days after femoral artery ligation.The results show that muscle small vessel densities were comparable between PD-1 KO and WT mice 7 days after femoral artery ligation,while the vessel density was significantly lower in PD-1 KO mice as compared with WT mice 14 days after femoral artery ligation.This result suggesting PD-1 KO attenuated muscle angiogenesis in mice after hindlimb ischemia.Since the hindlimb perfusion regulates the exercise capacity,we determined the mouse exercise capacity before ischemia,as well as 14 and 21 days after hindlimb ischemia.We found that the exercise capacity was not different between WT and PD-1^-/-mice under control conditions,but the exercise capacity of PD-1^-/-mice was significantly decreased as compared with WT mice at 14 and 21 days after femoral artery ligation,indicating PD-1-/-severely impaired mouse exercise capacity after hindlimb ischemia.Since the hindlimb perfusion regulates the muscle dystrophy and fibrosis after hindlimb ischemia.These results showed that the muscle fiber cross-sectional area was significantly decreased in PD-1^-/-mice as compared to WT mice 21 days after femoral artery ligation.In addition,PD-1^-/-resulted in a significant increase of muscle fibrosis in mice 21 days after femoral artery.The results suggested that PD-1-/-exacerbated mouse muscle dystrophy and fibrosis after hindlimb ischemia.2.PD-1^-/-exacerbated muscle inflammatory response and oxidative stress in mice after hindlimb ischemiaLeukocytes are almost undetectable in normal skeletal muscles obtained from either PD-1^-/-or WT mice.However,hindlimb ischemia caused increases of leukocyte infiltration inside myocytes and in the interstitial space in both WT and PD-1^-/-mice.Immunofluorescence staining further confirmed that CD45+leukocytes are significantly increased in gastrocnemius muscle in PD-1^-/-mice,as compared with WT mice,3 days after femoral artery ligation.Flow cytometry data further demonstrated that hindlimb ischemia resulted in a significantly greater increase of muscle macrophage infiltration in PD-1^-/-mice as compared with the WT mice.Macrophages appear to be the major leukocyte subset infiltrated in the skeletal muscle in both PD-1^-/-and WT mice after ischemia.These results suggest that PD-1-/-exacerbated the infiltrations of inflammatory cells in mouse muscle after hindlimb ischemia.We further determined reactive oxidative species?ROS?in gastrocnemius muscle by dihydroethidium staining.While the ROS contents of normal muscles were similar in PD-1^-/-and WT mice,however,the muscle intracellular ROS production was significantly increased in PD-1^-/-mice as compared with WT mice day 3 after hindlimb ischemia.The number of muscle ROS positive cells and the signal intensity in these ROS positive cells were both increased in PD-1^-/-mice as compared with WT.These results suggest that PD-1-/-exacerbated muscle oxidative stress in mice after hindlimb ischemia.We also determined mRNA contents of a group proinflammatory cytokines such as IL-1?,MCP-1,IL-6 and IFN-?etc.While hindlimb ischemia caused significant increases of muscle IL-1?,TNF-?,IL-6,MCP-1 and IFN-?mRNA in both PD-1^-/-and WT mice 3 days after femoral artery ligation,PD-1^-/-only caused significantly greater increases in skeletal muscle IFN-?.We further determined muscle IFN-?protein content in muscles with or without ischemia from both PD-1^-/-and WT mice,and the result demonstrated that PD-1^-/-significantly increased muscle IFN-?protein in muscle after ischemia3.IFN-?increased vascular endothelial cell apoptosis,and impaired proliferation,migration,tube formationIFN-?stimulation reduced the vascular tube formation capacity.In addition,IFN-?stimulation also significantly attenuated HUVEC migration as determined by scratch repair assay.IFN-?stimulation was significantly attenuated HUVEC proliferation.Furthermore,we found that IFN-?caused a significant increase in HUVEC apoptosis.4.PD-1^-/-enhanced muscle inflammatory cell produced IFN-?and TNF-?in mice after hindlimb ischemiaNotice the increased muscle IFN-?production in PD-1^-/-mice,we further determined IFN-?production in both CD45+leukocytes and CD45 negative cells in mice 3 days after FA ligation.We found that total IFN-?positive leukocytes,total IFN-?positive leukocytes/muscle weight,the percentage of IFN-?positive leukocytes and the mean signal intensity?MFI?of IFN-?positive leukocytes in CD45+subset was significantly increased in PD-1^-/-mice after FA ligation.Meanwhile,the percentage of IFN-?positive cells in CD45 negative cells?non-leukocytes?were almost undetectable in both wild type and PD-1-/-mice.These results suggested that PD-1-/-enhanced muscle leukocytes produced IFN-?in mice after hindlimb ischemia.In addition,as compare with corresponding wild type mice,total IFN-?positive macrophages,total IFN-?positive macrophages/muscle weight,the percentage of IFN-?positive macrophages and the MFI of IFN-?positive macrophages in total macrophages were both significantly increased in PD-1^-/-mice after FA ligation.Together,these findings indicate macrophages likely contribute to the increased muscle IFN-?production in PD-1^-/-mice after FA ligation.The total numbers of TNF-?positive leukocytes/muscle weight was significantly increased in PD-1^-/-mice after FA ligation,but as compared to corresponding wild type mice,the percentages and total number and MFI of TNF-?positive leukocytes were not increased in PD-1^-/-mice.Meanwhile,the percentage of TNF-?positive cells in CD45 negative cells was almost undetectable in both wild type and PD-1^-/-mice.The total numbers of TNF-?positive macrophages/muscle weight were significantly increased in PD-1^-/-mice after FA ligation,but as compared to corresponding wild type mice,the percentages and total number and MFI of TNF-?positive macrophages were not increased in PD-1^-/-mice.5.PD-1 blocking antibodies impaired blood flow recovery,angiogenesis,exercise capacity and exacerbated mouse skeletal muscle dystrophy and fibrosis after hindlimb ischemiaWe found that inhibition of PD-1 with blocking antibodies had no detectable effect on hindlimb blood perfusion in mice before ischemia.However,PD-1blocking antibodies significantly attenuated blood perfusion in mice 14 and 21 days after FA ligation.PD-1 blocking antibodies significantly attenuated muscle capillary density at21 days after hindlimb ischemia.These results indicating that PD-1 blocking antibodies impaired angiogenesis after hindlimb ischemia.In addition,PD-1 blocking antibodies significantly reduced the overall exercise capacity in mice 14 and 21 days after hindlimb ischemia.Moreover,we found that the muscle fiber cross-sectional area was significantly decreased in PD-1 blocking antibodies treatment mice as compared to WT mice 21 days after femoral artery ligation.In addition,PD-1 blocking antibodies treatment resulted in a significant increase of muscle fibrosis in mice 21days after femoral artery ligation.Conclusion:Our findings demonstrate that inhibition of PD-1,through genetic or pharmacologic means,exacerbated ischemia-induced tissue IFN-?production,leukocyte infiltration,oxidative stress,and tissue injury,as well as consequent reduced vessel regeneration in mice after femoral artery ligation.The IFN-?may come from inflammatory cells.In addition,IFN-?increased vascular endothelial cell apoptosis,and impaired proliferation,migration,tube formation.These observations indicate that PD-1 exerts an important role in regulating ischemia-induced angiogenesis through modulating the tissue inflammatory response.