Vascular Calcification Is Coupled With Phenotypic Conversion Of Vascular Smooth Muscle Cells Through KLF5Mediated Transactivation Of The Runx2Promoter

Vascular calcification is one of the major complications of atherosclerosis,diabetes mellitus, hypertension, and chronic kidney disease. It decreasesvessel elasticity and compliance of the vessel wall, leading to increasedincidence of many cardiovascular diseases, such as myocardial infarction andplaque rupture during atherosclerosis. Many factors, including abnormalmineral metabolism, chronic inflammation, and oxidative stress, are associatedwith the generation of vascular calcification. Accumulating evidence indicatesthat vascular calcification is not due to passive precipitation of calciumphosphate, but rather is a tightly regulated process that resembles theossification processes of the bone. The process includes phenotypicconversion of vascular smooth muscle cells (VSMC) to a phenotype withosteogenic characteristics. In response to high phosphate stimulus, VSMCsincrease expression a number of bone-associated proteins such as alkalinephosphatase (ALP), matrix Gla protein (MGP), osteopontin (OPN), andosteocalcin (OC) in vitro and in vivo，and decrease expression of VSMCdifferentiation marker genes including smooth muscle (SM) α-actin andSM22α.Recent studies have shown that Runx2, a key transcription factor thatregulates osteoblast and chondrocyte differentiation, is expressed inatherosclerotic calcified human vascular tissues and in calcifying aorticsmooth muscle cells in mice but not in normal vessels. Runx2deficiencyinhibits oxidative stress-reduced expression of VSMC marker genes anddecreases formation of osteoclast-like cells in the calcified lesions. Angiotensin II (Ang II) exacerbates the vascular calcification throughactivation of Runx2and NF-кB, supporting an essential role of Runx2inVSMC calcification.The transcription factors controlling the phenotypic state of VSMCs arealso involved in the pathogenesis of vascular calcification. For example, highphosphate induces KLF4expression in VSMCs. KLF4knockdown attenuateshigh phosphate-induced VSMC phenotypic switching into osteogenic cells, aswell as reduces expression of osteogenic genes and calcium deposition. KLF4is also induced markedly in the calcified aorta of adenine-induced uremic rats.Our previous studies showed that Ang II inhibits p21expression by inducingthe expression of KLF5，a pro-proliferative transcription factor, and promotesphenotypic conversion of VSMCs into proliferative and synthetic cells. KLF4and KLF5are closely related members of the KLF family of transcriptionfactors; a pair of positive and negative regulators of cellular proliferation.However, it is unknown whether KLF5exerts any effect on calcification ofVSMCs. In addition, the potential link between KLF5and Runx2-mediatedvascular calcification has not been examined. The aims of the present studieswere to elucidate the effect of KLF5on VSMC calcification in vitro and invivo and to investigate the actual relationship between KLF5and Runx2inmediating vascular calcification.Part one High phosphate induces phenotypic switching of vascularsmooth muscle cells into osteogenic cells and KLF5expressionObjective: To investigate the relationship between KLF5andosteoblastic transdifferentiation of VSMCs induced by high phosphate.Methods: Rat VSMCs were treated with3.8mM phosphate to inducecell calcification model. The degree of local calcium deposition, that includeAlizarin red S staining, calcium content, alkaline phosphatase (ALP) activity,were tested to reflect the osteogenic transformation. Then the expression ofKLF5, the osteogenic marker gene Runx2, the VSMC marker gene SM α-actinand SM22α were analyzed by real-time PCR and Western blotting. Ex vivoaortic ring mineralization with3.8mM phosphate were used to vertify these calfification change.Results: Alizarin red S staining showed obvious mineralizationnodules.The ALP activities and calcium content were also increased bytreament with3.8mM phosphate. VSMC calcification model was successfullyestablished. Obvious black calcium depositions were oberved in calcifiedaortic ring. The mRNA and protein level of KLF5and Runx2were increased,while SM α-actin and SM22α were decreased both in vitro and in ex vivoOur study showed that VSMC osteoblastic transdifferentiation wasinduced with high phosphate. KLF5and Runx2were activated during thecalcification process. The expressions of KLF5and Runx2were positivelycorrelated with the degree of VSMCs calcification.Part two KLF5regulates osteoblastic transdifferentiation of vascularsmooth muscle cellsObjective: To elucidate the effect of KLF5on VSMC differentiation intoosteoblast-like cells induced by high phosphate.Methods: We overexpressed KLF5with adenovirus and knocked downKLF5with special siRNA respectively to test the calfification degree ofVSMC by Alizarin red S staining and calcium content mesurement. Then theexpression of KLF5, Runx2, SM α-actin and SM22α were analyzed byreal-time PCR and Western blotting. We constructed the Runx2promoterreporter, and co-transfected VSMCs with the KLF5expression plasmid.Luciferase reporter assay was carried out to investigate if Runx2was the targetgene of KLF5. Chromatin immunoprecipitation (ChIP) and oligo pulldownwere used to test the binding site.Results: We found that KLF5overexpression did not cause detectablecalcification in VSMCs cultured with control medium as indicated by thecalcium deposition levels and Alizarin red S staining. However, when VSMCswere exposed to high phosphate medium, the calcium deposition in KLF5overexpression reached1.56-fold above the GFP adenovirus and themineralization nodules increased significantly. Furthermore, expression ofosteoblast marker gene Runx2increased1.2-fold and2.3-fold in VSMCs infected with Ad-GFP plus high phosphate and Ad-KLF5plus high phosphatetreatment when compared with untreated cells. In addition, the expression ofVSMC markers SM α-actin and SM22α was decreased by40%and30%respectively after VSMCs were exposed to high phosphate medium. To furtherverify this result, rat aortic rings were incubated in a medium with elevatedphosphate concentration and similar results were obtained in the ex vivomodel.Upon exposure to high phosphate, calcium deposition in VSMCsknocking down KLF5was decreased to30%of the cells transfected withscramble siRNA and the mineralization nodules decreased significantly..Additionally, knockdown of KLF5significantly decreased the expression ofosteogenic marker gene Runx2by70%in response to high phosphatestimulation, as indicated by real-time PCR and Western blotting.Knockdown of KLF5and incubation with high phosphate medium increasedthe expression of SM α-actin and SM22α when compared with the cellstransfected with scramble siRNA and then incubated with high phosphate.Luciferase assay showed that the Runx2promoter could be activated byKLF5overexpression in KLF5expression plasmid concentration-dependentmanner with High phosphate treated. ChIP assay showed that high phosphatemarkedly promoted the binding of KLF5to proximal region of the Runx2promoter (－185bp—－27bp), which contains KLF5-binding sites1～4; nosignificant binding of KLF5was detected when distal Runx2promoter regioncontaining KLF5-binding sites5～8was amplified. Consistent with the resultsof the ChIP assays, oligonucleotide pull-down assay showed that the bindingof KLF5to the sites1～2was increased more significantly than sites3～4byhigh phosphate treatment.Our study show that KLF5contributes to high phosphate induced VSMCosteoblastic transdifferentiation through transactivation of Runx2. The bindingsite for KLF5has been identified at－185～－27bp within Runx2. KLF5represents an important new regulator of osteoblastic transdifferentiation of VSMCs.Part Three The role of KLF5in vascular calcification in chronic renalfailureObjective: To examine the expression of KLF5and Runx2in thecalcified aorta of rats with chronic renal failure in vivo.Methods: Chronic renal failure was induced in SD rats by0.75%adenine-rich chow and serum biochemical parameters were analyzed usingautoanalyzer. Cardiac function was evaluated by using high-resolutionechocardiography. Doppler echocardiography, Von kossa staining and calciumcontent assay were used to determine the calcification; KLF5, Runx2and SMα-actin were analyzed by immunohistochemistry; real-time PCR and Westernblotting were used to examine the expression of KLF5and Runx2.Results: We confirmed the successful model of chronic renal failure bythe increased amount of creatinine, nitrogen, inorganic phosphate andcalcium-phosphate product. Cardiac dysfunction and heart failure wereinduced in rats by adenine-rich chow. Medial calcification was found in theaorta by colour Doppler echocardiography and Von Kossa staining. Thecalcium content was increased in the calcified aorta and serum phosphatecorrelated significantly with the aortic calcium content. The expression ofKLF5and Runx2was increased in calcified aorta, while SM α-actin wasdecreased in calcified aorta, which was consistent with the results in vitro.Both the expression of KLF5and Runx2was increased in the calcifiedaorta of adenine-induced ureic rats, which suggest the relation between KLF5and vascular calcification.Conclusions:1. High phosphate induces the expression of KLF5and VSMCcalcification.2. KLF5binds directly to the Runx2promoter and activates itstranscription. 3. KLF5is induced markedly in the calcified aorta of adenine-induceduremic rats.4. Vascular calcification is coupled with phenotypic conversion ofVSMCs through KLF5-mediated transactivation of Runx2promoter.