| Background Ascending thoracic aortic aneurysm(ATAA)is characterized by the enlargement of ascending aorta to the extent of 50% larger than normal ones due to aberrant structural alteration and is different from AA of other sites when concerning embryonic origin,tissue structure and pathological mechanisms.Since ATAA develops asymptomatically,it can be life-threatening due to rupture or dissection in advanced stage,if not be detected and managed early.While surgical replacement of diseased aorta is currently the only effective treatment,it costs much and imposes high operation risks and more complication probabilities on the old.Therefore it is meaningful to clarify the underlying mechanisms of ATAA development,in an effort to find a preferable substitute treatment.The pathological changes of dilated aorta wall is marked by cystic medial degeneration and destruction of extracellular matrix,such as fragmented elastin and collagen fibers.Over the past years,researchers have generally recognized that the phenotype switching of vascular smooth muscle cells(VSMCs)plays a pivotal role during the development of ATAA.Stimulated by various ouside factors,VSMCs usually undertake a transition from a contratile status to a synthetic one,during which long spindle-shaped VSMCs change into more squared ones.Meanwhile,synthetic and secretory activities enhance while contraction function weakens.Such smooth muscle cell specific proteins as α-SMA and SM22α are decrease in expression whereas extracellular matrix proteins like collagens,proteoglycans and some proteases are synthesized and secreted extensively.Among them are the increased and activated matrix metalloproteinases(MMPs)which destroy the extracellular matrix by degrading fibers and other constitutive proteins,finally resulting in aorta expansion.However the detailed molecular mechanisms of VSMCs phenotype switching have not been elucidated to date.micro RNAs(mi RNAs),a major kind of non-coding RNA with around 22 nt in length,are found involved in growth,carcinogenesis and development of cardiovascular diseases through targeting at various genes expression.Among them,micro RNA-29 family(mi R-29)played a vital role in fibrosis by regulating expression of such extracellular matrix proteins as collagens.Recent researches showed mi R-29 was abberently expressed in abdominal and thoracic aneurysm and yet the detailed expression and mechanisms are not currently known.Whether and how mi R-29 is involed in the development of ATAA have not yet been reported to the best of our knowledge.Objectives 1.To ascertain the expression of mi R-29 family in ATAA.2.To clarity the specific role of mi R-29 family in phenotype switching,proliferation and migrarition of VSMCs.3.To investigate the detailed mechanisms of mi R-29 involved in VSMCs phenotype switching.Methods 1.Tissue specimens collection and cell culture(1)Collection and pretreatment: Dilated ascending aortae were collected from ATAA patients during surgery.Patients with genetic diseases such as Marfan syndrome or with bicuspid aortic valve etc.were excluded.Ascending aortae from heart transplant donors or patients undergoing isolated aortic valve replacement were used as control.All specimens were cut into three pieces and emerged in formalin,liquid nitrogen and normal saline each for pathological examination,PCR and cell isolation,respectively.(2)Human VSMCs isolation: By using explant culture,aneurysmal and normal aorta medium were cut into pieces of 2 mm3 and then were attached onto the plate to let the VSMCs migrate outward under culture.(3)Construction of mouse model of ATAA: Mini pumps containing Ang II(1000ng/Kg/min)were implanted subcutaneously in C57BL/J mice for 28 days to generate aortic dilatation.Pumps containing normal saline were used as control.At week 0,1,2 and 4,ascending aorta diameters were mearsured echocardiographically,and certain number of mice were random Ly selected for harvest of ascending aorta.Similarly,harvested asending aortae were cut into three parts and were emerged in 4% paraformaldehyde,liquid nitrogen and normal saline respectively for further experiments.(4)Isolation of mouse VSMCs: medium of mouse ascending aorta were cut into pieces and digested with collagen II(3mg/m L)under 37℃ on a rotatable platform for 4 hours,then digested solution was filtered,centrifuged and resuspended with fresh medium in plate for culture of cell.(5)Identification of VSMCs:By using immunofluorescence staining,isolated cells were stained for VSMCs specific marker,α-SMA and SM22α.(6)Pathological staining: hematoxylin and eosin(H&E)staining was used to investigate the structural changes and Victoria blue staining for the manifestation of collagens and elastin fibers.MMPs and KLF4 expressions in tissue were detected by immunohistochemistry.2.The investigation of the roles and mechanisms of mi R-29 in VSMCs activities(1)q RT-PCR: Total RNA of tissue or cells was extracted using TRIzol method and mi R-29 expression was detected using q RT-PCR according the manual instructions.(2)In vitro cell model: PDGF-BB(10ng/m L)and Ang II(100m M)were respectively applied to stimulate the cells for at least 24 hours to iinduce the phenotype switching.(3)Transfection by liposome: The liposome of Lipo2000 was used to deliver the mi R-29 b mimic or KLF4 si RNA or HDAC10 plasmid into mouse VSMCs,according to the specifications.Culture medium was replaced 6 hours later after transfection.(4)Virus transfection: After determing the tite and MOI(multiplicity of infection),virus liquid was directly added into the culture medium to infect cells.(5)Western Blot: Total proteins of cells were extracted and homogenized in concentration,then Western blot was applied to detect the expression levels of certain proteins(α-SMA,SM22α,MMP-9,MMP-2,etc.).(6)ELISA: The concentration of MMP-9/2 in cultured medium was dedermined via using ELISA Kit.(7)CCK-8 assay: CCK-8 Kit was used to judge the proliferation activities of cells after treatment.(8)Wound healing assay: Cells were scratched departed to observe the migration ability after treatment.(9)Dural-luciferase reporter gene assay: After constructing the reporter plasmid(plasmid with wide type or mutated KLF4 3’UTR),plasmids were delivered by Lipo2000 into cells and substrates were added to detect the fluorescence intensity.(10)Ch IP-PCR: According to instructions of Ch IP assay kit,KLF4 binded DNAs were pull down and purified,then were amplified using PCR.(11)Agarose gel electrophoresis: 1%(w/v)agarose gel was prepared and amplified DNAs were loaded and electrophoresed under 60 V to separate DNA fragment.3.Statistic analysis Data were presented as mean±SD(standard deviation)or median(percentage).Student t test orχ2 test were used for two independent samples.All analysis were performed using SPSS software(Version 22.0)and p<0.05 was considered statistically significant.Results 1.mi R-29 b was downexpressed in ATAA accompanied by the upregulation of MMP-9/2.The results of pathological staining showed that the wall of aneurysmal aorta destroyed greatly and MMP-9/2 were stained intensely compared with normal aorta.PCR results indicated that mi R-29 b was downexpressed both in human aortic aneurysmal specimens and aneurysm originated VSMCs and its expression levels in aneurysmal tissue were reversely correlated with aneurysm diameters.However,there is no discrepancy concerning the expression of mi R-29 a and mi R-29 c.During the development of mouse ATAA,PCR found that mi R-29 a and mi R-29 b were both progressively downexpressed.Mi R-29 b also downexpressed in PDGF-BB or Ang II treated mouse VSMCs,accompanied by the increased expression of MMP-9/2 in culture medium in a time dependent manner,which suggest mi R-29 b may be related with MMP-9/2.2.Overexpression of mi R-29 b can inhibit the phenotype switching,proliferation and migration of mouse VSMCs and downregulate the expression and secretion of MMP-9/2.After mouse VSMCs were first stimulated either with PDGF-BB or Ang II and then transfected with mi R-29 b mimic,Western blot results showed that compared with scramble transfection,overexpression of mi R-29 b resulted in the elevated expression of such contractile markers as α-SMA and SM22α while in inhibition of OPN and MMP-9/2 expression.El ISA results also indicated the lower concentration of MMP-9/2 in cultured medium,when compared with cells of no overexpression of mi R-29 b.CCK-8 assay suggested a reduced proliferation activity of mi R-29 b transfected VSMCs and wound healing assay showed the overexpresson of mi R-29 b inhibited the migration of mouse VSMCs. Dural-luciferase reporter gene assay verified that mi R-29 b targeted directly at the 3’UTR of KLF4 m RNA.Immunohistochemistry showed KLF4 was highly expressed in aneurysmal tissue.Overexpression of KLF4 in mouse VSMCs resulted in enchanced migration activity but decreased proliferation,as demonstrated respectively by wound healing and CCK-8 assay.Inhibition of KLF4 after stimulation by Ang II in mouse VSMCs almost alleviated the downexpression of α-SMA and SM22α while reduced the level of MMP-9 and OPN,but no effect on MMP-2 expression,as shown by western blot.Similarly results were gained through resuce experiment,i.e.,overexpresson of mi R-29 b after overexpression of KLF4 in mouse VSMCs.Those together suggested that KLF4 was negatively involved in the effects of mi R-29 b on VSMCs phenotype switching.Ch IP assay further illustrated that KLF4 binded to the promoter regioun of HDAC10.Overexpresion of HDAC10 secondary to overexpression of KLF4 in mouse VSMCs,as the rescue experiment suggested,restored the expression ofα-SMA but significantly reduced MMP-9 expression,with no effect on MMP-2 expression.3.mi R-29 b can partly reverse phenotype switching of mouse VSMCs and inhibit the associated MMP-9 expression through targeting at KLF4.Conclusions mi R-29 b is downexpressed in tissues of ATAA and in process of phenotype switching of VSMCs.Overexpression of mi R-29 b can partly reverse phenotype switching of VSMCs and inhibit the expression and secretio of MMP-9 through directly targeting at KLF4 and HDAC10 as the downstream of KLF4 may participate in the process. |
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