CD226 Deficiency In Platelet Diminishes Positive Effects Of Platelet-Rich Plasma (PRP) Treatment In Knee Osteoarthritis In Mice

Background:Knee osteoarthritis（OA）in the elderly is a common multifactorial bone and joint disorder with complex pathogenic factors and serious impact on function.Current conservative treatment for OA is predominantly the temporary relief of pain and local swelling,but do little to rescue joint cartilage degeneration.In recent years,platelet rich plasma（PRP）,which widely used in tissue regeneration area,has been used into the OA treatment field.PRP core technology is high-platelet ratio blood product produced by patient blood gradient centrifugation injected into the joint.Studies have shown that multiple bioactive factors secreted from platelet,such as growth factors and anti-inflammatory factors,are benefit for the repairment of osteoarthritis and cartilage regeneration,and safe and effective in the treatment of knee joint injury.Meta-analysis and genome-wide association studies indicated that the adhesion molecule CD226 had regulatory effects in the process of megakaryopoiesis and platelet formation.This study aimed to investigate the mechanism of CD226 modulate platelet function,thus affecting PRP treatment in a mouse model of OA.Objective:The purpose of this work was to assess the role of CD226 in the function of megakaryocytes（MKs）/platelets,thus evaluating whether the adhesion molecule CD226affects the efficiency of intraarticular injection of PRP in ameliorating joint damage caused by surgical destabilization of the medial meniscus（DMM）-induced knee OA in mice.Methods:1.Platelet specific knockout CD226 mice and OA model of DMM mice were constructed.Platelet specific knockout CD226 mice were constructed by site-specific recombinant enzyme system Cre/Lox P and CRISPR/Cas9 technique,and the knockout efficiency was verified.C57BL/6 mice were used to establish surgical destabilization of the medial meniscus（DMM）model of OA.To observe the effect of PRP treatment,C57BL/6 mice were randomly divided into 5 groups:A sham-operated group,DMM+saline group,DMM+HA group,DMM+PRP from CD226^fl/fl mice and DMM+PRP from CD226^fl/flPF4-Cre mice.Safranin O-fast green staining and Osteoarthritis Research Society International（OARSI）-modified Mankin scores were used to grade articular cartilage degeneration.2.The effect of CD226 on platelet function.The serum concentration of PDGF-AB and platelet factor 4（PF4）were analyzed by ELISA.Routine blood tests and transmission electron microscopy were applied to observe platelet characteristics and ultrastructure of platelets between CD226^fl/fl PF4-Cre and littermate mice.Flow cytometric analysis was used to determine immature/mature percentage of platelets.HE staining was used to observe the histological alterations of mice spleen and bone marrow MKs.3.The effect of CD226 on platelet autophagy.Western blot and immunofluorescence staining were used to detect the autophagy molecule expression levels in MKs/platelets and Dami cells.4.Proteomics analysis the differential gene expression after platelet CD226 knocked out and then verify.The i TRAQ-based proteomics analysis was performed to explore the differential expressed proteins in CD226 deficient platelets and littermate platelets.Results with Q value<0.05 and fold change>1.2 are set as the significant threshold for differential expression.Western blot and immunofluorescence staining were used to verify the results of the i TRAQ analysis.5.Further observe the effect of PDGF-AA supplementation on PRP function after platelet CD226 knockout.C57BL/6 mice were randomly divided into 4 groups:DMM+PRP from CD226^fl/flmice,DMM+PRP from CD226^fl/flPF4-Cre mice,DMM+PRP from CD226^fl/flmice+PDGF-AA and DMM+PRP from CD226^fl/flPF4-Cre mice+PDGF-AA.Safranin O-fast green staining and OARSI scores were used to grade articular cartilage degeneration.Results:1.Base on Western blot and flow cytometry,it was confirmed that platelet specific knockout CD226 mice with high knockout efficiency were successfully constructed.Intraarticular saline and HA injection group showed more severe cartilage defects and massive osteophyte formation than the sham group.But the treatment of the PRP from CD226^fl/fl mice significantly alleviated the cartilage defects,which could be reversed by PRP from the CD226^fl/fl PF4-Cre mice.2.We found that the whole structure of platelets in the CD226^fl/fl PF4-Cre mice was intact,and area of platelet was also similar to that of control mice.However,average area ofα-granule per platelet was significantly reduced in CD226^fl/flPF4-Cre mice than that in littermates CD226^fl/fl mice.Both concentration of PDGF-AB and PF4 in PRP were significantly reduced in CD226^fl/flPF4-Cre mice compared with CD226^fl/fl mice.These results indicated that deficiency of CD226 may affectα-granule secretion in platelet.Platelet counts and PCT were significantly reduced in CD226^fl/flPF4-Cre platelet than that of in littermates by routine blood test.The HE staining results showed that CD226^fl/flPF4-Cre mice have significantly increased MK counts in BM of the femur;however,CD226^fl/flPF4-Cre mice have significantly decreased MK counts in spleen compared with CD226^fl/flmice.The above results suggested a hyperactivity status of central immunological organ may compensate the dysfunction of MKs/platelets in the peripheral immune system.The ratio of TO⁺immature platelets was significantly decreased in CD226^fl/flPF4-Cre mice.3.Beclin 1 and LC3 were less abundant in CD226^fl/flPF4-Cre platelets than in CD226^fl/flplatelets.CD226 deficiency in platelets resulted in up-regulation of P-m TOR,while the total protein level of m TOR was not changed.Like the results in platelets,CD226knockdown significantly decreased autophagy in Dami cells.Immunofluorescence labeling analysis demonstrated that Beclin 1 was appeared in a lower level in MKs in CD226^fl/flPF4-Cre mice than that in CD226^fl/flmice.4.Based on the combined results of proteomics,GO,and KEGG pathway analyses,we selected PDGF-A,PDGF-B and S6 ribosomal protein for further validation in vitro.A Western blot was performed and the results showed that the deficiency of CD226 in platelets decreased the level of PDGF-A,PDGF-B and S6 ribosomal protein expression.Immunofluorescence labeling also showed that S6 ribosomal protein was lower in MKs in CD226^fl/flPF4-Cre mice than that in CD226^fl/flmice.5.Pathological OARSI scores,osteophyte size and maturity demonstrated PDGF-AA could restore CD226 deficiency-caused impaired protective effect of PRP in DMM mice.Conclusion:1.Platelet CD226 deficiency may affect megakaryocyte/platelet homeostasis and interfere with platelet development and secretion.CD226 deficiency in platelet leads to the increase of MKs in central immune organs and less MKs in peripheral spleen;the number of platelets in circulation is reduced,and the proportion of immature platelets is decreased.2.Platelet CD226 deficiency can lead to decreased ribosome expression,CD226deficiency leads to decreased autophagy and ribosome biogenesis in MKs/platelets,which may attenuate the homeostasis of MKs/platelets.3.Platelet CD226 deficiency can lead to decline the protective effect of PRP in DMM-induced knee OA in mice,aggravate the cartilage injury of the knee joint in mice,and then affecte the cartilage repair effect of PRP on the mouse OA model,whereas PDGF-AA could largely reverse this decline in vivo.