Mechanism Of Inhibition Of Restenosis After Vascular Injury By AGGF1 Via Interaction With Integrin α7

AGGF1 is a new Angiogenic Factor with G-patch and FHA domains 1 identified during the early study of rare disease Klippel-Trenaunay syndrome(KTS).AGGF1 can promote endothelial cell proliferation and angiogenesis by activating the AKT signaling pathway,which plays a key role in vein development.Later,AGGF1 mutations were found to cause not only KTS,but also cancer,and AGGF1 expression level was abnormal in mouse models or patient tissues for common diseases such as coronary heart disease and heart failure.In animal models,direct injection of purified AGGF1 protein has a good therapeutic effect on coronary heart disease,heart failure and peripheral arterial disease.In a vascular injury model,AGGF1 protein expression level was decreased,and direct injection of purified AGGF1 protein inhibited intimal neoplasia and restenosis after vascular injury by maintaining the contractile phenotype of vascular smooth muscle cells.However,the molecular mechanism of AGGF1 in the treatment of restenosis after vascular injury needs to be further studied.More importantly,the receptor of AGGF1 protein on vascular smooth muscle cell surface has not been identified,and the key region of the AGGF1 protein regulating smooth muscle phenotypic switching remains to be identified.In this study,the AGGF1 protein was systematically truncated.The AGGF1 protein has 714 amino acids,and a total of 13 mutant proteins from AGGF1-C1 to AGGF1-C13 were obtained by truncating AGGF1 at every 50 amino acids from the C-terminus.The expression of vascular smooth muscle cell contraction markers MYH11,α-SMA and SM22 was detected by RT-PCR and Western blot analyses.It was found that AGGF1-C1 and AGGF1-C2 mutants and wild type AGGF1 had the effect of maintaining the contractile phenotype of vascular smooth muscle cells,but AGGF1-C3 to AGGF1-C13 mutant proteins lost this effect.These results suggest that AGGF1-mediated regulation of smooth muscle phenotypic switching depends on amino acids 564-614 of AGGF1.Protein-cell adhesion assays also showed that the adhesion of the AGGF1 protein to smooth muscle cells was dependent on amino acids 564 to 614.By analyzing the amino acid sequence of this region,we found that this region contains a sequence motif of RDDAPAS,which is highly homologous to the RGD sequence(a polypeptide sequence composed of arginine,glycine and aspartic acid that can bind to a variety of integrin proteins specifically).The main function of the RGD sequence motif is to mediate extracellular signals to regulate cell proliferation and differentiation by binding to integrin family proteins.Therefore,it is speculated that integrin family proteins may be the receptors of AGGF1 on vascular smooth muscle cells.To test this hypothesis,two integrin family members(integrin α7 and integrinα8)with high expression in vascular smooth muscle cells were investigated as candidates for the AGGF1 receptor.Co-immunoprecipitation and protein-cell adhesion assays showed that integrin α7,but not integrin α8,interacted with AGGF1 and mediated AGGF1 adhesion to vascular smooth muscle cells,suggesting that Integrin α7 may be the receptor of AGGF1 on vascular smooth muscle cells.Western blot analysis showed that knockdown of integrinα7 in vascular smooth muscle cells inhibited the effect of AGGF1 on the expression of contractile marker proteins(MYH11,α-SMA and SM22)in vascular smooth muscle cells,and attenuated the effect of AGGF1 on the inhibition of the MEK-ERK-ELK signaling pathway.In mice,the inhibitory effect of AGGF1 on intimal neoplasia and restenosis after vascular injury was eliminated by lentiviral knockdown of integrin α7 expression,indicating that the AGGF1 protein inhibits intimal neoplasia and restenosis after vascular injury through integrin α7.Subsequently,in order to investigate the key amino acids responsible for the interaction between AGGF1 and integrin α7 in the RDD region of the AGGF1 protein,the AGGF1 RDD motif was mutated to alanine(A)point by point.Through coimmunoprecipitation and cell proliferation and migration experiments,it was found that AGGF1-ADD mutant could not interact with integrin α7,and lost its ability to inhibit the migration and proliferation of vascular smooth muscle cells.These results indicate that the first R of the RDD motif is critical for the AGGF1-integrin α7 interaction,and this interaction is essential for AGGF1 to maintain the vascular smooth muscle contractile phenotype.Finally,in this study,the wild-type mice were subjected to carotid artery injury and intraperitoneal injection of wild type AGGF1 or truncated AGGF1 mutants.The function of neointima and vascular smooth muscle cells after vascular injury was detected by immunohistochemistry,and it was found that the full-length wild type AGGF1 and AGGF1-C1 and AGGF1-C2 mutants could inhibit neointimal neointima and restenosis after vascular injury,and increased protein expression of contractile markers in vascular smooth muscle cells.However,AGGF1-C3 and AGGF1-ADD mutants lost these effects,indicating that the RDD motif of AGGF1 interacting with integrin α7 plays an important role in inhibiting intimal neogenesis and treating restenosis after vascular injury.In conclusion,this study suggests that integrin α7 may be a receptor for AGGF1 on the surface of vascular smooth muscle cells.AGGF1 maintains the contractile phenotype of vascular smooth muscle cells through integrin α7 and inhibits intimal neoplasia and restenosis after vascular injury in mice.It was found that AGGF1 inhibits the MEKERK1/2-ELK signaling pathway,and intimal neoplasia and restenosis after vascular injury,and regulates vascular smooth muscle cell functions depending on the RDD sequence motif of AGGF1 that interacts with integrin α7.These results further expand our understanding of the function of AGGF1,and identify a new molecular mechanism by which AGGF1 regulates the phenotypic transition,proliferation and migration of vascular smooth muscle cells,and inhibits intimal neoplasia and restenosis after vascular injury.Meanwhile,these results further establish the AGGF1 protein as a novel target to develop therapeutic strategies for the prevention and treatment of blood vessel restenosis after interventional therapy.