PGCA Promotes The Development Of Aortic Dissection By Regulating Smooth Muscle Cell Function Via P38MAPK Signaling Pathway

Objective:Aortic Dissection(AD)is a life-threatening vascular disease in thoracic and cardiac surgery,sharing similar risks with acute cerebral hemorrhage and myocardial infarction,and is associated with a very poor prognosis.It is one of the primary causes of sudden clinical death.At present,there are no specific diagnostic biochemical indicators and therapeutic drugs,and the pathophysiological mechanism is still unclear.This study intends to explore the role of PGCA in the occurrence and development of aortic dissection and the related possible mechanisms from human and mouse,in vitro and in vivo,gene and proteins in multiple dimensions and dimensions.Methods:1.From Jan.2021 to Dec.2022,a total of 10 normal aortas and 15 dissected aortas,along with their corresponding blood samples,were collected from organ donors and patients diagnosed with acute aortic dissection.Animal model of aortic dissection in mice was established by administration of β-aminopropyl amine(BAPN)in drinking water into C57BL/6 mice.All tissue were verified by HE and EVG staining.RT-qPCR,Western blot and immunofluorescence staining were used to detect the expression levels of ACAN and its encoded protein PGCA in murine and human normal and dissected aorta,as well as the distribution of PGCA protein in aortic dissection tissues.2.Lentivirus transfection technique and recombinant PGCA protein were used to reconstruct an environment for contractile HASMCs,in which PGCA was inhibited and increased in extracellular matrix(ECM),respectively.The expression level of ACAN was verified by RT-qPCR,and the concentration of PGCA in extracellular matrix was detected by Elisa.Cell proliferation capacity was detected by CCK8 and Ed U experiments,cell apoptosis level was detected by flow cytometry and TUNEL experiments,cell migration and invasion capacity were detected by scratch assay and Transwell experiments,and the cell phenotypic transformation was detected by signature protein via Western blot,respectively.3.To investigate how different levels of PGCA modulates function of HAVSMCs,RT-qPCR and Western blot were used to detect the phosphorylation levels of the signature proteins of p38 MAPK and NF-κB signaling pathways in in four different concentrations of PGCA in HAVSMCs.SB203580,a specific pathway inhibitor of p38 MAPK,was used to confirm a regulatory role of PGCA in p38 MAPK signaling pathway.4.Adeno-associated virus(AAV2-sh-Acan)were constructed to specifically knock down Acan in aortic vascular smooth muscle.Mice were randomly assigned into three groups and received different treatments: 1.AAV2-sh-Acan(tail vein injection of AAV2-sh-Acan),2.vector(tail vein injection of AAV2 carrying an empty vector),and 3.mock(injection of saline).The mice in the first two groups were received BAPN treatment as previously described to induce aortic dissection.Aortic tissues were collected and subjected to RT-qPCR,Western blot,HE staining,and elastic fiber staining analysis.The severity of aortic dissection was compared by calculating the rupture point of elastic fiber.Finally,the survival curve and the incidence of aortic dissection were calculated among three groups.Results:1.BAPN-containing water feeding successfully induced aortic dissection in mice.The HE and EVG staining found broken elastic fibers in aortic dissection patients and mice,confirming the existence of aortic dissection.RT-qPCR and Western blot confirmed a higher expression level of ACAN and PGCA in both murine and human dissected aortas.Immunofluorescence staining showed that PGCA was mainly concentrated in the extracellular matrix and smooth muscle cells.2.Compared to control,PGCA was successfully knockdown by lentivirus sh-ACAN in both HAVSMCs and culture medium.I found that HAVSMC apoptosis was reduced in cells with a low level of PGCA in ECM,but increased in cells with a high level of PGCA in ECM.Conversely,PGCA inhibition enhanced HAVSMC migration and invasion capabilities,while addition of the recombinant PGCA protein into the cultured HAVSMCs inhibited cell migration and invasion.Additionally,I found PGCA knockdown upregulated contractile HAVSMC marker SMA,but downregulated synthetic HAVSMC marker OPN.An opposite effect was observed in HAVSMCs treated with recombinant PGCA protein.3.It was found that p65 and p38 phosphorylation levels were significantly decreased by PGCA inhibition,but increased by addition of PGCA protein into the cultured HAVSMCs.Additionally,the regulatory effects of PGCA on p38 phosphorylation were reversed by SB203580.4.No death was found in the mock group.The expression levels of PGCA in the aorta of mice treated with AAV2-sh-Acan were significantly lower than that of mice treated with the empty vector and saline.Consequently,longer survival time,lower aortic dissection incidence,and fewer rupture points were observed in mice treated with AAV2-sh-Acan.Conclusion:This study first verified that PGCA is highly expressed in aortic dissection diseases,and the main expression sites are extracellular matrix and VSMC.PGCA can affect the functional changes of smooth muscle cells,including proliferation,apoptosis,migration and invasion,and cell phenotype changes.In smooth muscle cells,PGCA can activate phosphorylation of the p38 MAPK signaling pathway.PGCA can also promote aortic dissection in vivo.This study suggests that PGCA may play a key role in the occurrence and development of aortic dissection and is expected to become a target for the diagnosis and treatment of aortic dissection.