Perturbed RDNA Transcription Induces Vascular Smooth Muscle Cell Senescence And Formation Of Aneurysm-like Lesions

ObjectiveSenescence of vascular smooth muscle cells may play a pivotal role in the pathogenesis of various vascular diseases.Aneurysm is a pathological dilation of a blood vessel lumen caused by degeneration of the blood vessel wall.A common clinical aneurysm is Abdominal Aortic Aneurysm(AAA).The total mortality caused by AAA rupture and its related complications exceeds 80%.The latest research found that the aging of VSMCs is closely related to the occurrence and development of AAA.Cell senescence is a sequela of chronic insults by various stress conditions,among which oxidative stress,genotoxic stress,and replicative stress are well established examples.The nucleolus is the site where ribosomal DNA(rDNA)transcription and ribosome biogenesis occur.Inhibiting rDNA transcription(mediated by RNA polymerase I or Pol I)in eukaryotic cells triggers a unique cellular stress response known as nucleolar stress(also termed ribosomal stress).The previous research of our group found that inhibiting rDNA transcription and inducing nucleolar stress in vascular smooth muscle cells can cause G2/M cell cycle arrest,and the expression of certain cell senescence markers is up-regulated.In this topic,we conduct a more in-depth study of this phenomenon.We propose the following scientific hypothesis:Perturbing rDNA transcription to induce nucleolar stress can induce the senescent phenotype of vascular smooth muscle cells,and can accelerate the appearance of aneurysm-like degenerative pathological changes in the arterial wall.In order to confirm this hypothesis,we mainly focused on the following three scientific issues:(1)Whether the expression of the key components of the Pol I transcription complex in human AAA tissues and mouse AAA models has changed;(2)Utilizing smooth muscle-specific TIF-IA-deficient mice and a specific Pol I inhibitor(CX-5461)as a means to study whether perturbation of rDNA transcription and induction of nucleolar stress can accelerate the occurrence and development of aneurysm-like degenerative disease in the blood vessel wall(3)Clarify whether nucleolar stress response induced by perturbed rDNA transcription accelerates the senescent phenotype of vascular smooth muscle cells,explore possible mechanisms,and further verify whether these cytological changes above exist in AAA tissues.Methods?.Changes in expression of the key components of Pol ? transcription complex in aneurysm tissues and the effect of perturbed rDNA transcription on the occurrence and development of aneurysm-like degenerative lesions1.Animal models and groupingSelect ApoE-/-male mice aged 8 weeks to establish Ang ?-induced AAA models.Select C57BL/6 male mice aged 8 weeks to establish CaCl2/PBS(Ca/P)-induced AAA models.ApoE-/-mice were randomly divided into 2 groups:sham operation and Ang?-induced AAA model.On the 7th day and the 28th day,tissues were taken from each group.C57BL/6 mice were randomly divided into 2 groups,PBS control and Ca/P AAA model.On the 7th day and the 28th day,tissues were taken from each group.In the drug intervention experiment,C57BL/6 mice were randomly divided into 3 groups,namely PBS group,Ca/P group and CX+Ca/P group.2.Angiotensin ?-induced AAA modelAng ? was injected into the osmotic pump at a dose of 1000 ng/min/kg,and the sham operation(Sham)group was injected with the same amount of normal saline.ApoE-/-mice were anesthetized by intraperitoneal injection of 1%sodium pentobarbital(40mg/kg)solution,and the mice were placed in prone position.The neck was prepared and a transverse incision of about 1 cm long was taken,and the back skin was bluntly freed with the subcutaneous tissue,place the osmotic pump under the skin and suture the incision layer by layer.After the mice regained consciousness,they were put back into the squirrel cage and given normal diet.Samples were taken on the 7th and 28th days to detect UBF,TIF-IA,TBP,RPA43(Pol ? and TIF-?A connected subunit)mRNA expression changes in vascular tissues by qPCR.3.CaCl2/phosphate-induced AAAC57BL/6 mice were anesthetized by inhaling isoflurane gas with a small animal anesthesia machine.After skin preparation,a median abdominal incision was taken to find the abdominal aorta,and the surrounding connective tissue was carefully separated to expose the sub-renal abdominal aorta segment.Cover the surface of the abdominal aorta with a tampon fully soaked in CaCl2 solution(0.5M)and incubate for 10 minutes.Then change to a cotton swab soaked in sterile phosphate buffered saline(PBS)and continue to incubate for 5 minutes.After spreading and incubating,flush the abdominal cavity with normal saline,and blot the flushing liquid with a sterile cotton ball.In the PBS control group,only PBS-soaked cotton slivers were applied for 15 minutes.The tissues were sutured layer by layer,and the mice were put back into the squirrel cage after waking up.Meloxicam(1mg/kg)was given for analgesic treatment.Samples were taken on the 7th and 28th day to detect UBF,TIF-IA,TBP and RPA43 mRNA changes in the vascular tissues by qPCRDrug intervention experiment:In the CX+Ca/P group,50?L of Pluronic F127 solution containing CX-5461(Final concentration:10?M)was added to the place where the cotton sliver was applied.In the PBS group and Ca/P group,the same amount of Pluronic F127 solution was added dropwise,and the intestinal tube was reset after it became gel-like.Four weeks later,samples were taken to observe whether the aortic blood vessels had obvious bulging and measure its maximum diameter.The tissue sections were stained with elastic fiber staining(Verhoeffs van gieson staining,EVG staining)and hematoxylin-eosin(HE)staining.The destruction of the vascular elastic layer was scored and compared.4.Breeding and phenotype detection of smTIF-IA-/-miceMating female TIF-IAflox/flox mice and male Cre(SMA-Cre)miceto obtain TIF-IA+/--Cre mice,and then male and female TIF-IA+/--Cre mice were mated and reproduced to create smooth muscle-specific TIF-IA-deficient mice(smTIF-IA-/-).Littermate TIF-IA+/+ mice were used as wild type controls.Observe the status and body weight of the mice,detect and record the blood pressure of the mice.Take mice of different ages for anatomy,treat the aortic vessel with EVG and HE staining and count the average thickness of the vascular media.Perform immunohistochemical staining to detect smooth muscle alpha actin(?-SMA),matrix metalloproteinase(MMP)-2,MMP-9 and macrophage marker F4/80.5.Collection and processing of human abdominal aortic aneurysm tissue specimensTissue specimens of patients who underwent abdominal aortic aneurysm resection and artificial blood vessel replacement at Shandong Provincial Hospital from October 2016 to September 2018 were collected.Part of the residual normal abdominal aorta tissue during kidney transplantation performed at the Organ Transplant Center of Shandong University Qilu Hospital from November 2016 to March 2018 was collected as a normal control.The mRNA levels of UBF,TBP,TIF-IA and the ratio of 45S pre-rRNA/mature 18S rRNA in the tissues were detected by qPCR.We also used Western blot assay to detect the protein levels of TBP and TIF-IA in the tissues.II.The effect of perturbed rDNA transcription on vascular smooth muscle cell senescence and its relationship with aneurysm lesions1.Effect of Pol I inhibitor on the senescent phenotype of vascular smooth muscle cellsTwo Pol I inhibitors CX-5461 and BMH-21 were used to treat mouse smooth muscle cell lines(MOVAS)and human aortic smooth muscle cells(HASMC),respectively.Senescence-associated ?-galactosidase(?-Gal)assays was used to detect the ratio of senescent cells,and Real-time qPCR was used to detect the expression changes of senescence-related pathway proteins p21cip1,p161NK4a,plasminogen activator inhibitor-1(PAI-1)and interleukin IL-6,IL-8.2.Effect of TIF-IA gene silencing on the senescent phenotype of vascular smooth muscle cellsUtilizing lentiviral vectors expressing shRNA to knockdown TIF-IA gene in MOVAS cells and Real-time qPCR assay was used to detect the efficiency of lentiviral interference.To clarify the effect of TIF-IA gene knocking down,we use Real-time qPCR to detect the key components of Pol I transcription complex UBF,polr-1A(encoding the largest subunit of Pol I)and RPA43 and some key factors including p21Cip1,PAI-1 and IL-6,IL-8.After knocking down TIF-IA gene,Cell Counting Kit-8(CCK-8)was used to detect changes in cell proliferation,and flow cytometry was used to detect changes in cell cycle.3.Effect of TIF-IA gene silencing on DNA damage response in vascular smooth muscle cellsAfter knocking down the TIF-IA gene,perform immunofluorescence assay to detect the distribution of nucleophosmin(NPM)in MOVAS cells,and also utilize Western blot and immunofluorescence assays to detect p53 protein and its phosphorylation level and ATR phosphorylation level,respectively.Besides,utilize immunofluorescence to detect the expression of histone H2AX phosphorylation(yH2AX)and DNA-dependent protease catalytic subunits(DNA-PKCs)in the nucleus of MOVAS.Alkaline comet electrophoresis was utilized to detect the DNA breaks.4.The changes of nucleolar stress,cell senescence and DNA damage response in AAA tissuesReal-time qPCR was used to detect the expression of p21Cip1,PAI-1,MMP-2,MMP-9 in human AAA tissues.Immunohistochemistry/immunofluorescence analysis was used to detect the phosphorylation levels of p53,ATR and ATM/ATR substrate S/T*Q related to DNA damage pathway in human AAA tissues.Perform immunofluorescence analysis to detect the expression of yH2AX in vascular media of human AAA tissues by co-localizing yH2AX and a-SMA.In order to clarify whether DNA damage in AAAs is related to increased nucleolar stress,immunofluorescence staining was performed on NPM,and NPM and yH2AX were co-localized for further analysis.Immunohistochemistry analysis was performed to detect the phosphorylation levels of p53 and ATR in aorta media of smTIF-IA-/-mouse.Senescence-associated?-galactosidase(?-Gal)assay and deoxyribonucleotide terminal transferase-mediated nick end labeling assay(TUNEL)were performed to detect the senescence and apoptosis of SMCs in the aorta of smTIF-IA-/-mouse.Immunohistochemistry analysis was performed to detect the expression of PAI-1 and IL-8.ResultsI.Changes in expression of the key components of Pol I transcription complex in aneurysm tissues and the effect of perturbed rDNA transcription on the occurrence and development of aneurysm-like degenerative lesions1.Changes in expression of the key components of Pol I transcription complex in human AAA tissuesReal-time qPCR analysis showed that,as compared to normal aortas,human AAA tissues exhibited a downregulation in the expression of TIF-IA,whereas the level of TBP was upregulated.The expression of UBF was not changed.Western blot analysis showed that the level of TIF-IA protein was downregulated while TBP exhibited a downregulation.Besides,the ratio of 45S pre-rRNA to mature 18S rRNA was decreased.2.Changes in expression of the key components of Pol I transcription complex in Ang II-induced AAA tissuesDoppler ultrasound analysis showed that the maximum diameter of blood vessels in Angiotensin ?-induced AAAs increased by 50%on the 7th day,and the maximum diameter of the blood vessels increased by 140%on the 28th day.The expression levels of UBF,TBP and RPA43(the TIF-IA interacting subunit of Pol I)were not altered at day 7,while the level of TIF-IA was reduced.At day 28,expressions of UBF and TIF-IA were downregulated.In comparison,the expressions of TBP or RPA43 were not significantly altered at either time points3.Changes in expression of the key components of Pol I transcrintion complex in Ca/P-induced AAA tissuesHE staining assay showed that the AAA model induced by Ca/P began to undergo elastic fiber destruction in vascular media and inflammatory cell infiltration at day 7;the elastic fibers in vascular media were damaged and fractured severely,and inflammatory cell infiltration was obvious at day 28.In the abdominal aorta from C57BL/6 mice with Ca/P-induced AAA,the expression of UBF was not changed at day 7 but was decreased significantly at day 28.The expression of TIF-IA was significantly downregulated at both day 7 and day 28.The expression of TBP was up-regulated at day 7,but there was no significant change at day 7.In contrast,the expression of RPA43 did not change significantly at either time point4.Formation of spontaneous aneurysm-like lesions in the aortic wall of smTIF-IA-/-micesmTIF-IA-/-mice were viable at birth,but showed growth retardation,with the body weight being approximately 10-20%less than that of littermate wild type controls.Spontaneous mortalities were observed between 4 to 10 weeks.The death was occurring without obvious preceding behavioral abnormalities.We performed autopsies for these mice,but did not find bleedings in the abdominal or thoracic cavity Histopathological examinations revealed that 100%of smTIF-IA-/-mice harbored wide spread aneurysm-like lesions in both of the thoracic and abdominal aortasHistopathological analysis revealed that the vascular lesions exhibited derangement and fragmentation of the elastic lamina,and diminished SMCs Immunohistochemistry analysis demonstrated that smTIF-IA-/-aortas exhibited increased levels of matrix metalloproteinase(MMP)-2 and MMP-9.Moreover,there was an increase in macrophage accumulation in the perivascular region of smTIF-IA-/-aorta.5.Specific Pol I inhibitor CX-5461 aggravates Ca/P-induced AAA formationTo further clarify whether perturbed rDNA transcription had a pathogenic role in AAA formation,we utilized the selective Pol I inhibitor CX-5461.We demonstrated that perivascular treatment with CX-5461 aggravated the development of AAA induced by CaCl2/phosphate in wild type mice.CX-5461 increased the incidence of AAA formation following CaCl2/phosphate treatment.The average diameter of CX-5461-treated abdominal aortas was significantly greater than that of controls Histopathological analysis revealed that in CX-5461-treated aortas,elastic lamina fragmentation and loss of medial SMCs were more prominent.Moreover,we assessed the severity of elastic lamina destruction using an Elastin Score as described.It was shown that the Elastin Score was significantly higher in AAAs with CX-5461 treatment.?.The effect of perturbed rDNA transcription on vascular smooth muscle cell senescence and its relationship with aneurysm lesions1.Inhibition of rDNA transcription induces senescence-like phenotype in smooth muscle cellsCX-5461 treatment of MOVAS cells increased the number of senescent cells as revealed by ?-Gal staining.Moreover,qPCR analysis showed that CX-5461 and BMH-21 treatment upregulated p21Cip1,p16INK4a and PAI-1,as well as markers of the senescence-associated secretory phenotype including IL-6 and IL-8.To further corroborate the results from MOVAS cells,we treated human aortic smooth muscle cells with BMH-21.It was demonstrated that BMH-21 increased the number of?-Gal-positive cells;and both of BMH-21 and CX-5461 increased the expression levels of p21Cip1,p16INK4a and PAI-1.Western blot experiments also confirmed the increased p21Cip1 expression in BMH-21-treated human smooth muscle cells.2.TIF-IA gene silencing induces senescence-like phenotype in MOVAS cellsValidation experiments demonstrated that TIF-IA shRNA transduction diminished the expression of TIF-IA by 90%,but did not affect other Pol I co-factors including UBF,RPA43 or Polrl A.SMCs with TIF-IA knockdown proliferated slower than control cells as revealed by CCK-8 assay.Flow cytometry analysis showed that TIF-IA gene knockdown induced G2/M phase block and S phase prolongation in cells.Consistently,TIF-IA knockdown also increased the levels of PAI-1,p21Cip1 IL-6 and IL-8 compared with control3.Perturbed rDNA transcription induces nucleolar stress and p53 pathway activation in smooth muscle cellCellular immunofluorescence analysis showed that knocking down TIF-IA gene reduced the ratio of nucleolar protein NPM in the nucleolus/nucleoplasm in MOVAS cells,suggesting that NPM diffused from nucleolus to nucleoplasm.The nucleolus morphology changed,and nucleolar caps appeared.The structure suggested that the cell underwent a nucleolar stress response.Western blot and immunofluorescence analysis revealed that perturbed rDNA transcription significantly increased the phosphorylation level of p53(Ser1 5),while the total p53 protein level did not change.4.The phosphorylation of p53 caused by perturbed rDNA transcription may be related to the activation of non-canonical DNA damage responseCellular immunofluorescence and/or Western blot analysis showed that knocking down TIF-IA in MOVAS cells significantly increased the phosphorylation level of ATM/ATR,and increased the ratio of ?H2AX and DNA-PKCs positive cells as well However,alkaline comet assay found that perturbed rDNA transcription did not cause widespread DNA strand breaks,suggesting that the p53 phosphorylation caused by perturbed rDNA transcription in vascular smooth muscle cells may be related to the activation of non-canonical DNA damage response.5.The correlation between nucleolar stress,DNA damage response,aging and aneurysm lesionsqPCR showed that the expressions of PAI-1,p21Cip1,MMP-2 and MMP-9 were all upregulated in human AAA tissues,indicating an increase in cell senescence.To clarify whether AAA was associated with increased nucleolar stress,we performed immunofluorescence staining for NPM.It was found that the number of cells with aberrant nucleoli morphology(exhibiting diffused NPM fluorescence signal)was increased in AAA tissues.Increased p53 accumulation has been observed in human AAA tissues by immunohistochemistry analysis.To corroborate these results,we performed yH2AX staining in aortic sections,and demonstrated that the prevalence of cells exhibiting positive yH2AX staining in the nuclei was significantly increased in AAA tissues.Immunofluorescence analysis demonstrated that the level of phospho-ATR was also increased in the medial layer of AAA.Moreover,the phosphorylation level of the ATM/ATR substrate S/T*Q motif was augmented in AAA tissues.Performing double immunofluorescence labeling for yH2AX and NPM,we found that over half of the yH2AX-positive cells showed co-localization of the yH2AX and NPM signals.On the other hand,most of the ?H2AX-positive cells showing no ?H2AX-NPM co-localization had intact nucleoli.These data suggested that in AAA tissues,the phosphorylation level of p53 is accompanied by the increase of nucleolar stress and DNA damage response,and the incidence of DNA damages within the nucleolar compartment may involve rDNATo further confirm our findings in the smTIF-IA-/-model,we performed immunohistochemical staining,and demonstrated that phospho-p53 and phospho-ATR levels were all elevated in the tunica media of TIF-IA-/-aorta.?-Gal staining confirmed that the number of senescent cells was also increased in smTIF-IA-/-aortas.Furthermore,smTIF-IA-/-aortas showed increased levels of IL-8 and PAI-1 in the tunica media as compared to wild type controls.However,we found that there was not a significant difference in the number of apoptotic cells in the media between wild type and smTIF-IA-/-models.ConclusionThe expression of the key components of the Pol I transcription complex in aneurysm tissues changed significantly.On the whole,perturbed rDNA transcription in SMCs accelerated the occurrence and development of aneurysm-like degenerative lesions.In vitro,perturbing rDNA transcription in SMCs and inducing nucleolar stress may activate the p53 pathway by triggering a non-canonical DNA damage response,which further led to senescence-like phenotypes in SMCs.In human AAA tissues,the phosphorylation level of p53 is accompanied by the increase of nucleolar stress and DNA damage response,and the incidence of DNA damages within the nucleolar compartment may involve rDNA.Innovation and significance1.It was the first time that perturbed rDNA transcription may be involved in the occurrence and development of aneurysms2.The created smooth muscle-specific TIF-IA-deficient mice may be used as a new animal model for studying the occurrence of aneurysm-like lesionsLimitations1.In vivo,intervention experiments for cell senescence had not been completed.2.In vitro,we did not observe the effect of inhibiting DNA damage response on the senescence of SMCs,and could not establish the causal relationship between the induced non-canonical DNA damage response and cell senescence.