The Therapeutic Effect Of AGGF1 On Thoracic Aortic Aneurysms And Underlying Molecular Mechanism

Thoracic aortic aneurysm refers to the irreversible local or diffuse outward dilation of the thoracic aorta induced by various causes,resulting in"tumorous"enlargement.This dilation is more than 1.5 times of the size of the normal vascular diameter.Currently,the clinical treatment of TAA mainly relies on surgery and lacks effective drug therapy.AGGF1（Angio Genic factor with G-patch and FHA domains 1）is a novel angiogenic factor but also a key determining factor of the cell fate.AGGF1 regulates the differentiation of hemangioblasts,multi-potent stem cells that can differentiate into blood cells and the cells of blood vessels,and affects the proliferation and migration of vascular smooth muscle cells（VSMCs）and other cell functions.Because VSMCs abnormalities play an important role in the pathogenesis of TAA,and AGGF1 regulates the function of VSMCs,this study hypothesized that AGGF1 affected the development of TAA by regulating the function of VSMCs.Three different TAA-related animal models were used in this study,including Transverse Aortic Constriction（TAC）-induced aneurysm model,genetic Fbn1^C1041G/+mouse（genetic TAA model associated with Marfan syndrome）TAA model,and an TAA mouse model induced byβ-aminoproprorile（BAPN）.In this study,we investigated the role and molecular mechanism of AGGF1 in TAA and its therapeutic potential through in vivo experimental studies with the three different types of TAA mouse models and in vitro cellular and molecular studies.The main results are as follows.（1）The expression level of AGGF1 was significantly decreased in the diseased aortic tissues of TAA patients and three TAA animal models.The aortic tissue samples of TAA patients and non-TAA patients were used for immunohistochemical staining with an anti-AGGF1 antibody.Results showed that AGGF1 levels were significantly decreased in the diseased aortic tissues of TAA patients（P<0.001）.Similar results were obtained form three TAA animal models.（2）Transforming growth factorβ1（TGF-β1）signaling pathway was enhanced,and TAA-related phenotypes were aggravated by AGGF1haploinsufficiency.A TAA model was established by generating high biomechanical pressure in the right carotid artery and ascending aorta through TAC,resulting in vascular dilation,remodeling,and inflammation.Doppler echocardiography results showed that compared with wild-type mice,the TAC-induced TAA phenotypes,including aortic dilation and remodeling,inflammation and TGF-β1 signaling were aggravated in Aggf1^+/-mice or smooth muscle cell-specific Aggf1 knockout mice.（3）The enhanced transforming growth factorβ1（TGF-β1）signaling pathway and TAA-related phenotype were effectively reversed by intraperitoneal injection of recombinant AGGF1 protein in three TAA animal models.First,In this TAA model,intraperitoneal injection of AGGF1 protein（5μg/10g）effectively inhibited TGF-β1signaling and TAC-induced aortic dilation,remodeling,and inflammation,but the mutant AGGF1 protein with a mutated RDD motif(AGGF1-RDD^del)did not have these effects.Second,we found that in Fbn1^C1041G/+mice,the ascending aorta was spontaneously dilated and remodeled with aging.Doppler echocardiography showed that intrapitoneal injection of AGGF1 protein effectively inhibited the dilation of the ascending aorta and medium thickening,vascular remodeling,and inflammatory response associated with TAA and TGF-β1 signaling in Fbn1^C1041G/+mice.Third,the TAA model was induced by feeding mice with 0.25%BAPN drinking water for 28 days.Doppler echocardiography showed that BAPN significantly induced ascending aorta dilatation in mice,and reduced the survival rate of mice.AGGF1 protein effectively reduced the ascending aorta dilatation,remodeling,inflammation,and TGF-β1 signaling,and increased the survival rate of mice.（4）A series of molecular and cellular experiments were used to dissect the molecular mechanism of AGGF1-mediated inhibition of TGF-β1 signaling.We found that AGGF1inhibited LAP-TGF-β1 cleavage,prevented TGF-β1 maturation,and reduced Smad2/3 and ERK1/2 phosphorylation levels through its interaction with integrinα7,a VSMCs surface receptor for AGGF1.In addition,we found that TGF-β1 inhibited AGGF1 expression,TGF-βinhibitor pirfenidone（PFD）promoted AGGF1 expression,forming a negative feedback cycle.In mice treated with PFD,vascular dilation,remodeling,and inflammation after TAC were inhibited in wild-type mice,but not in Aggf1^+/-mice,suggesting that the inhibitory effect of TGF-β1 inhibitor depends on high AGGF1 expression level.In conclusion,this study found that the AGGF1 expression level was significantly decreased in TAA patient aortic samples and three different TAA mouse models,and the decreased AGGF1 expression level promoted the development of TAA,indicating that AGGF1 played an important role in the development of TAA.We also found that AGGF1effectively inhibited TAA-related phenotypes by inhibiting TGF-β1 signaling in a RDD domain dependent manner.Studies on the molecular mechanism of AGGF1 not only found that AGGF1 inhibited the downstream signaling pathway of TGF-β1,but also found for the first time that binding of integrinα7 to LAP-TGF-β1 inhibited TGF-β1 maturation;AGGF1 promoted the binding of integrinα7 to LAP-TGF-β1 in an RDD dependent manner.This study for the first time found the therapeutic potential of AGGF1 on TAA and the underlyuing molecular mechanism,suggesting that AGGF1 is a drug target for the treatment of TAA.