The Effect And Mechanism Of CREG In Abdominal Aortic Aneurysm

Objective:Abdominal aortic aneurysm（AAA）is defined as permanent aortic dilation or bulging^[1].Epidemiology shows that AAA is very common in the population,and its rupture can lead to potentially life-threatening conditions^[2-4].Since 2000,statistics from Latin America and some high-income Asia-Pacific countries have shown that the prevalence of AAA has increased^[5,6].In the United States,deaths caused by AAA rupture rank 13th among all causes of death in the country,and it is estimated that it can cause about 15,000 deaths each year^[7].In China,studies have shown that after the age of 65,the incidence of AAA increases by 6%every ten years.In addition to gender,currently known risk factors include smoking,hypertension,and past history of myocardial infarction and peripheral arterial disease^[8].As our country gradually enters into an aging society,the increase in the above risk factors has led to a significant increase in the incidence of AAA,which has brought a heavy burden to social and economic development.At present,there is no treatment strategy that can prevent the progression and rupture of AAA,and endovascular or open surgical repair seems to be the only feasible method.Therefore,exploring mechanism of AAA onset and development is an urgent problem in this field.The pathological mechanism of the occurrence,progression and final vascular rupture of AAA has not been fully elucidated.It is generally believed that pathological changes such as inflammation infiltration,extracellular matrix（ECM）destruction,and vascular smooth muscle（vascular smooth cell,VSMCs）apoptosis plays an important role in the occurrence and development of AAA,among which VSMCs phenotype switch is the current hot research topic^[9-11].VSMCs phenotype transition refers to the transformation of VSMCs from contractile phenotype to synthetic phenotype when it is stimulated by pathological factors.Previous studies have shown that in the case of hypertension,atherosclerosis or other vascular diseases that may cause inflammation,the contractile phenotype of VSMCs restricts inflammation to the outer membrane to limit proteolysis.With the continuous action of pathogenic factors,VSMCs changes from a contractile phenotype to a secretory phenotype.Proteolytic enzymes（such as MMP2）which have the effect of degrading elastin and collagen and are an important factor leading to the destruction of the tunica media and the formation of aneurysms are secreted^[12].The cellular repressor of E1A-stimulated genes（CREG）is a transcription-related regulatory factor cloned from the Hela cell c DNA library of cervical cancer.It was first reported by Professor Gill from Harvard Medical School in 1998^[13].The c DNA of the human CREG gene is 663bp in length and consists of 220 amino acids^[14].Previously,Veal et al.introduced CREG protein into teratoma cells for in vitro culture and found that CREG not only inhibits cell proliferation,but also promotes the spontaneous differentiation of teratomas into nerve cells,suggesting that CREG may be a homeostatic regulator that induces cell differentiation^[15].Professor Yaling Han and her team conducted serial studies on the role of CREG in the cardiovascular system.The results are as follows:First,CREG protein began to express in embryonic vascular monolayer endothelial cells on embryonic day 9.5（E9.5）,and began to express in VSMCs and pericytes on day E10.5until the vascular matures^[16].During this process,CREG plays an important role in the biological regulation of embryonic vascular cell development and maturation.Second,under the action of various pathological factors such as vascular injury,the expression of CREG in vascular tissue is down-regulated^[17,18].Third,CREG gene overexpression can promote VSMCs differentiation,inhibit their apoptosis,proliferation and migration^[19].Fourth,CREG is an important regulator of vascular remodeling caused by hypertension^[20].In short,CREG plays an important role in maintaining vascular homeostasis and is expected to become a new target for prevention and treatment of AAA.This study aims to explore the role of CREG in the formation and development of AAA.By using mass spectrometry and other biochemical techniques,we expect to elucidate the specific mechanism and target of CREG in the pathogenesis of AAA,hoping to provide a novel experimental basis for the prevention and treatment of AAA.Methods:1.Establishment of AAA mouse model Methods:Male 8-week-old Apo E^-/-mice were implanted subcutaneously with osmotic mini-pump containing angiotensin Ⅱ（AngⅡ）to construct AAA model mice.The control group was given normal saline as control.Small animal ultrasonography was used to detect the maximum diameter of the mouse abdominal aorta and aneurysm formation at 1w,2w,3w,and 4w.Blood pressure were measured by tail-cuff method at time points mentioned above.The mice were sacrificed under anesthesia on day 28,then the abdominal aorta and aneurysm were collected.Masson’s trichrome staining was performed to evaluate fibrosis and EVG staining to evaluate elastic fiber degradation.Immunohistochemical（IHC）staining was used to detect CREG,α-SMA and MMP2/9 protein expression and location in cells or tissues.Western blot and quantitative PCR were used to detect CREG,α-SMA and MMP2/9 protein levels and m RNA levels in cells and tissues respectively.2.Detect the expression of CREG in the abdominal aorta during the formation of AAA in Apo E^-/-mice Methods:Male 8-week Apo E^-/-mice were used to establish AAA model mice by subcutaneously implanting AngⅡ,and sacrifice them under anesthesia on 0d,3d,7d,14d,and 28d respectively.Western blot was performed to detect the changes in CREG expression during the formation of AAA.3.Observe the effect of exogenous CREG in the development of AAA Methods:Male 8-week Apo E^-/-mice were used to construct AAA model by subcutaneously implanting AngⅡ.Each group has 30 mice and they were treated with Saline,AngⅡ or recombinant His-CREG protein.Thus,forming 3 groups including:Saline group,AngⅡ group,AngⅡ+His-CREG group.Small animal ultrasonography was used to detect the maximum diameter of the mouse abdominal aorta at 1w,2w,3w,and 4w and determine the rate of aneurysmal formation.Blood pressure were measured by tail-cuff method at time points mentioned above.On day 28,the mice were sacrificed under anesthesia,and the specimens were collected.Masson staining was performed to evaluate fibrosis and EVG staining to evaluate elastic fiber degradation.IHC staining was used to detect protein expression and location in cells or tissues.Western blot and quantitative PCR were used to detect the protein levels and m RNA levels in cells and tissues respectively.4.Clarify the role of endogenous CREG in the formation and development of AAA Methods:After genotype identification of Tg-CREG mice with Apo E^-/-background generating from the company,male mice and their littermate control mice were selected.AAA model were constructed by subcutaneous implantation of AngⅡ as described above.The following two groups were formed,i.e.AngⅡ group and AngⅡ+Tg-CREG group.5.Clarify the effect of CREG on VSMCs derived MMP2 production induced by AngⅡ Methods:The IHC staining was used to detect the expression of MMP2 in the abdominal aorta of mice in the CREG and/or AngⅡ treated groups.6.Clarify the correlation between CREG and MMP2 in VSMCs under AngⅡ stimulation Methods:For in vitro study,AngⅡ（1μM）was used to mimic AAA model in vivo.Western blot was used to detect the expression of CREG and MMP2 at 0h,24h,36h,48h after VSMCs were stimulated by AngⅡ.The time point which maximal changes occurred was chosen as the best stimulation time according to the results.7.Determine whether CREG can regulate MMP2 in vitroMethod:VSMCs were transfected with Ad-CREG to achieve overexpression of CREG（Ad-GFP was used as control）.Then AngⅡ（1μM）stimulation was given after grouping to form the following 4 groups:Ad-GFP,Ad-CREG,AngⅡ+Ad-GFP,AngⅡ+Ad-CREG.The changes in MMP2 expression were detected by Western blot.8.Clarify role of CREG in regulating MMP2 expression is at the transcription level or at the protein level in vitro Methods:VSMCs were transfected with Ad-CREG to achieve overexpression of CREG（Ad-GFP in the control group）.Then AngⅡ（1μM）or Saline was given after grouping to form the following 4 groups:NS+Ad-GFP,NS+Ad-CREG,AngⅡ+GFP,AngⅡ+Ad-CREG.The expression of MMP2 were detected by q RT-PCR.9.Screening the proteins that interact with CREG Method:Previous studies in our laboratory performed the experiment to screen the proteins that interact with CREG.In that experiment,recombinant human CREG protein with His tag was added to VSMCs cells.Proteins bound to CREG were obtained by immunoprecipitation technology.Then mass spectrometry analysis technology was used to detect the component（provided by the company）.10.Identification of the potential protein that regulates the expression of MMP2 and interacts with CREG according to the results of mass spectrometry analysis Methods:Search for transcription factors of MMP2 that have already been reported.Compare those with the results of mass spectrometry and identify the potential transcription factors that regulate MMP2.11.Clarify the relationship between YY1 and AAA Methods:Western blot was used to detect the expression of YY1 in the aortic tissue of AAA mice and control mice.12.In vitro experiments clarify the change of YY1 expression in the cytoplasm when stimulated by AngⅡ Methods:Mouse VSMCs were stimulated with AngⅡ（1μM）and collected at 0h,24h,48h,72h.The expression of YY1 in the cytoplasm was detected by Western blot.13.In vitro experiments clarified the changes in cytoplasm and nucleus of YY1 when stimulated by AngⅡ Methods:Mouse VSMCs were stimulated with AngⅡ（1μM）and collected at 0h,24h,48h,and 72h.The expression of YY1 in the cytoplasm and nucleus was detected by immunofluorescence.14.In vitro experiments clarify whether CREG has the effect of preventing YY1reduction in cytoplasm under AngⅡ stimulation Methods:Mouse VSMCs were transfected with adenovirus Ad-CREG to overexpress CREG,then stimulated with AngⅡ.The protein expression of YY1 in the cytoplasm was detected by Western blot.15.In vitro experiments clarify whether CREG could prevent YY1 nuclear translocation under AngⅡ stimulation Method:Mouse VSMCs were transfected with Ad-CREG to achieve overexpression of CREG（Ad-GFP in the control group）.Then treated with AngⅡ（1μM）or PBS after grouping to form the following 4 groups:NS+Ad-GFP,NS+Ad-CREG,AngⅡ+Ad-GFP,AngⅡ+Ad-CREG.The expression of YY1 in the cytoplasm and nucleus was detected by fluorescence and Western blot.16.Clarify the specific binding site of CREG and YY1 Methods:First,mouse VSMCs were transfected with Ad-YY1 and Ad-CREG for 24h,and cytoplasmic proteins were collected for Co-Immunoprecipitation（Co-IP）.CREG antibody was used for immunoprecipitation and YY1 antibody was used for Western blot analysis.Second,mouse VSMCs were transfected with Ad-YY1 and Ad-CREG for 24h,then cytoplasmic proteins were collected for Co-IP experiment.YY1 antibody was used for immunoprecipitation and CREG antibody was used for Western blot analysis.Third,primary VSMCs are transfected with Ad-YY1,and the CREG peptide,His-N100 was added for Co-IP experiment.His antibody was used for immunoprecipitation and YY1antibody was used for Western blot analysis.Forth,VSMCs were transfected with Ad-YY1,and the CREG peptide,His-C91 was added to perform Co-IP experiment,His antibody was used for immunoprecipitation and YY1 antibody was used for Western blot analysis.Results:1.The expression of CREG decreased in the mouse aortic tissue of AAA model in a time-dependent manner after AngⅡ stimulation,suggesting that the expression level of CREG was negatively correlated with the occurrence and development of AAA.2.Exogenous administration of recombinant CREG protein can inhibit AAA caused by AngⅡ stimulation.3.Endogenous overexpression of CREG can inhibit AAA caused by AngⅡ stimulation.4.In vivo,CREG can inhibit the expression of MMP2 in VSMCs induced by AngⅡ stimulation.In vitro,overexpression of CREG can inhibit the increase of MMP2expression in VSMCs induced by AngⅡ.5.In vivo,overexpression of CREG can prevent the reduction of YY1 expression in cytoplasmic proteins in VSMCs induced by AngⅡ.In vitro,overexpression of CREG can prevent YY1 nucleus translocation in VSMCs induced by AngⅡ.6.CREG binds to YY1.More specific,the N-100 peptide of CREG binds to YY1.Conclusion This study preliminarily explored the role and mechanism of CREG in the formation and progression of AAA.The results suggested that CREG exerts protective effect on AAA through down-regulation of MMP2 transcription via preventing YY1 nuclear translocation.