| Vascular adventitia has been indicated as the principal “injury-sensing tissue” withinthe artery, as it is capable of responding to different stimuli in an “outside-in” manner byoriginating and coordinating changes that progress toward the intima and finally end up invascular remodeling. As the major cell component in the adventitia, the adventitiafibroblasts (AFs) play a pivotal role in vascular remodeling owing to its remarkableplasticity. Once activated by diverse stimulation, the AFs undergo phenotypic changesending up in myofbroblasts (MFs) differentiation. Thus, these cells have contractileproperties and have a marked increase in proliferative and synthetic activities, migrating tothe intima and contributing to the vessel reshaping.Cellular repressor of E1A-stimulated genes (CREG) is ubiquitously expressed inmature tissues and cells in mammals and is expressed at very low levels in immature cells.It antagonizes the cellular transformation activities of primary cultured rat kidney cells, andpromotes human embryonic carcinoma cell differentiation even in the absence of aninducer. The results from our laboratory demonstrate that CREG participates in themaintenance of quiescent mature vascular smooth muscle cell (VSMC) phenotype in thearterial media by promoting VSMC differentiation and grow arrest and inhibiting migrationand apoptosis. Taken together, CREG plays a critical role in keeping cells or tissues in amature, homeostatic state.The mitogen-activated protein kinase (MAPK) pathways lead to a wide range ofcellular responses, including growth and differentiation. In mammals, three major MAPKpathways have been identified: ERK, JNK, and p38-MAPK. We have found that CREGplays a key role in modulating VSMC apoptosis through blocking the JNK and p38-MAPKsignal transduction pathways. In addition, the expression of CREG improves cardiacfunctions and inhibits cardiac hypertrophy through blocking ERK-dependent signaling. Therefore, MAPK pathways are involved in the CREG-mediated bio-functions in diversecell types.The hypothesis we propose here is that genetical modification with CREG gene in AFsmight inhibit the activation of AFs following vascular injury. These AFs over-expressingCREG might be more capable of resisting transdifferention into MFs, proliferation andmigration towards the lumen, as well as adventitial thickening. In view of theseconsiderations, we sought to evaluate the role of CREG gene expression in the modulationof AF phenotype transition and to delineate the mechanism by which CREG may confer itseffects. Our approach was to utilize a combination of established cell model and animalmodel, in which AFs were induced to transdifferentiate into MFs both in vitro and in vivo.The findings will highlight that adventitial CREG gene delivery may provide an efficientway for the treatment of artery remodeling related vascular disorders.The major results are presented below:1. The association of CREG protein with AF differentiation.Double immunofluorescent staining was performed with tissue sections from mousecarotid arteries to evaluate the relationship between the expression of CREG and α-SMA ininjured arteries, especially in the adventitia. Results demonstrated that normal mousecarotid arteries had little α-SMA expression throughout the tunica adventitia. Arteries atday1post-injury exhibited significantly higher immunofluorescence of α-SMA comparedwith that in non-injured arteries. Alpha-SMA expression was highest on day3, and beganto decrease on day7and progressively declined on day14. In contrast, immunofluorescentstaining revealed that CREG was expressed in the adventitia of normal arteries. Expressionof CREG in the adventitia of injured arteries was decreased on the1st day, reached itslowest value on the3th day, and increased gradually from the7th day, and was highercompared with that in non-injured arteries on the14th day after injury. Interestingly, thesedata indicate that the changes in CREG expression correlate with AF phenotypicmodulation in the adventitia of injured arteries, and that the injury-induced CREGdown-regulation may facilitate AF transdifferentiation into MFs and lesion formation.Angiotensin II (Ang II) is a potent inducer that prompts phenotypic transition of AFsinto MFs. AFs were cultured with medium in the presence of different concentrations (0,10nM,100nM,1μM and10μM) of Ang II for24h or in the presence of1μM Ang II for different times (0,12,24,48,72,96and120h). The data showed that expression ofα-SMA in AFs was very low, and Ang II remarkably increased expression levels of α-SMAin a dose-dependent and time-dependent manner, indicating that AFs acquire phenotypicfeatures of MFs after Ang II stimulation. Immunoblotting demonstrated that CREGexpression was decreased by45.5%and75.8%with1μM (p <0.01) and10μM (p <0.01)of Ang II (24h) treatment, respectively. The effect of Ang II on CREG expression was alsotime-dependent. CREG expression was decreased after24h of Ang II (1μM) treatment andreached a peak at120h, in decrements of31.5%,70.7%,90.5%,98.2%and98.9%at24h(p <0.01),48h (p <0.01),72h (p <0.01),96h (p <0.01), and120h (p <0.01),respectively. To elucidate which Ang II receptor was implicated in the induction of AFdifferentiation, the Ang II type1receptor (AT1R)-selective antagonist EMD66684(10μM)and Ang II type2receptor (AT2R)-selective antagonist PD123319(10μM) were used. Thepresence of EMD66684blocked the Ang II (1μM,24h)-induced changes of α-SMA andCREG protein expressions in AFs. In contrast, inhibition of the AT2R did not significantlyalter the changes of α-SMA and CREG protein expressions in response to Ang II. Theabove results suggest that CREG is associated with phenotypic modulation of AFs inducedbyAng II.2. CREG over-expression decreases Ang II-induced phenotypic modulation of AFs.To determine the role of CREG in AFs transition to MFs, we examined the effect ofCREG on α-SMA expression. The cells were incubated in the absence and presence of AngII (1μM) for24h. Immunocytofluorescence showed that α-SMA was not detectable in theabsence of Ang II. In contrast, AFs acquired phenotypic features of MFs after Ang IItreatment, showing an intense α-SMA expression. CREG-transduced AFs treated with AngII showed decreased transformation to MFs compared with non-transduced andGFP-transduced AFs treated with Ang II. In addition, similar to the immunocytofluorescentresults, western analysis demonstrated a similar trend in the expression of CREG andα-SMA. The protein expression of CREG was significantly higher in the control group thanthat in the Ang II group (128.9%±7.6%, n=3, p <0.05), and it was higher in theAd-CREG+Ang II group than that in the Ad-GFP+Ang II group (175.6%±4.8%, n=3,p <0.01). The protein expression of α-SMA was significantly lower in the control groupthan that in the Ang II group (13.9%±2.3%, n=3, p <0.01), and it was lower in the Ad-CREG+Ang II group than that in the Ad-GFP+Ang II group (21.6%±4.7%, n=3, p<0.01). Therefore, CREG stimulation inhibited the phenotypic switch of AFs andcounteracted the effects of Ang II.Phenotypic switching of AFs to MFs was accompanied by accelerated proliferationand migration. A proliferation assay performed with and without Ang II for12days showedthat the cell number of AFs in the four groups was unchanged for all treatments on the1stday. A significant decrease in the number of CREG-transduced cells was observed after2days (66.7%±9.7%, p <0.05, n=3),3days (63.3%±7.1%, p <0.01, n=3),4days(71.2%±5.4%, p <0.01, n=3) and5days (77.5%±6.6%, p <0.01, n=3) compared withnon-transduced and GFP-transduced AFs treated with Ang II. This finding indicatedinhibited cell proliferation, while no significant changes were observed after7to12days.The expression of CREG was increased on the2nd day after transient transduction (197.6%±33.5%vs. control, p <0.05, n=3), and it reached its highest value on the3rd day(482.7%±90.6%vs. control, p <0.01, n=3). CREG expression was gradually restoredafter4days (269.6%±52.4%vs. control, p <0.01, n=3), consistent with its inhibitory rolein proliferation. Further assessment of the proliferative activity of AFs was performed by aBrdU incorporation assay and FACS analysis. There was a3-fold reduction in BrdUincorporation in CREG-over-expressing AFs compared with untreated and GFP-expressingcells induced by Ang II (p <0.01, n=3). Moreover, CREG over-expression resulted insignificant retention of AFs in the G0/G1phase compared with the untreated andGFP-transduced cells (114.9%±5.5%, p <0.05, n=4).We then performed a wound healing assay to determine the effect of CREGover-expression on AFs migrating across the wound edge into the scratched area.Twenty-four hours after stimulation of AFs with1μM of Ang II, migration into thecell-free scratched area was observed. Representative microscopic images clearly showedthat CREG over-expression reduced AF migration induced by Ang II into the scratched areacompared with Ang II treatment alone. There was a significantly reduced number ofmigrating cells when CREG was over-expressed in AFs (66.0%±3.7%vs. Ad-GFP+AngII group, p <0.01, n=3). As an independent measure of AF migration, a transwellmigration assay was performed using a chamber coated with a thin layer of extracellularmatrix. Similar to the wound healing assays, over-expression of CREG substantially inhibited the migratory capacity of AFs induced by Ang II, as indicated by a markeddecrease in the number of migrated cells (54.2%±7.1%vs. Ad-GFP+Ang II group, p <0.01, n=3). In summary, CREG overexpression caused a significant attenuation of AFmigration induced by Ang II.3. CREG depresses Ang II-induced AF transformation, proliferation and migration bythe p38MAPK signaling pathway.We investigated whether ERK1/2, JNK1/2and p38-MAPK are involved inCREG-mediated regulation of Ang II-induced AF transformation, proliferation andmigration. We found that phosphorylation of p38-MAPK was significantly increased in theAng II group compared with that in the control group (279.1%±35.6%, p <0.01, n=3),and it was decreased in the Ad-CREG+Ang II group compared with the Ad-GFP+Ang IIgroup (56.1%±8.3%, p <0.01, n=3). However, phosphorylation of JNK1/2(104.3%±7.5%, p=0.27, n=3) and ERK1/2(101.2%±5.5%, p=0.73, n=3) were unchanged withCREG-transduced AFs after Ang II stimulation, suggesting that p38-MAPK may act as aregulator in the effect of CREG on AF function.Two additional series of experiments, in which inhibitors and activators of p38-MAPKwere applied, were performed to directly confirm this hypothesis. In the first series ofexperiments, AFs were pretreated with the p38-MAPK inhibitor SB203580(10μM) for1h,followed by stimulation with Ang II (1μM) for23h. First, findings of Western-blotanalysis showed that treatment of cells with SB203580inhibited Ang II-inducedupregulation of α-SMA (Ang II+SB203580group vs. Ang II group:42.2%±11.6%, p <0.01, n=3). Notably, CREG-transduced AFs treated with SB203580did not furtherdecrease the expression of α-SMA (Ad-CREG+Ang II+SB203580group vs. Ad-GFP+Ang II+SB203580group:91.3%±4.2%, p=0.41, n=3). Second, pretreatment of AFswith10μM SB203580markedly reduced CREG over-expression-mediated suppression ofBrdU incorporation (Ad-CREG+Ang II+SB203580group vs. Ad-GFP+Ang II+SB203580group:83.3%±11.5%, p=0.11, n=3). Third, the transwell migration assayshowed that CREG over-expression could significantly inhibit AF migration induced byAng II. This effect was markedly attenuated by pretreatment of cells with SB203580(Ad-CREG+Ang II+SB203580group vs. Ad-GFP+Ang II+SB203580group:90.3%±6.8%, p=0.25, n=3). In the second series of experiments, we first infected AFs with an adenovirus vectorcarrying constitutively active p38αMAPK (p38α CA) that results in p38-MAPK activation.AFs were then transiently transfected with another adenovirus vector carrying CREG.Transfected cells were treated with or without1μM Ang II. As seen in cells treated withSB203580, we found that p38α CA significantly attenuated CREG-mediateddown-regulation of α-SMA expression in AFs (Ad-CREG+Ang II+p38α CA group vs.Ad-GFP+Ang II+p38α CA group:101.5%±10.7%, p=0.13, n=3), suppression ofBrdU incorporation (Ad-CREG+Ang II+p38α CA group vs. Ad-GFP+Ang II+p38αCA group:95.1%±5.2%, p=0.29, n=3), and inhibition of AF migration (Ad-CREG+Ang II+p38α CA group vs. Ad-GFP+Ang II+p38α CA group:101.6%±4.1%, p=0.33,n=3).In conclusion, this study indicates that CREG expression was inversely correlatedwith the activation of SMC contractile protein α-SMA in AFs both in vivo and in vitro.Adenovirus-mediated CREG overexpression inhibited Ang II-induced AF transformationinto MFs, proliferation and migration. Moreover, the p38-MAPK signaling pathway wasinvolved in CREG-mediated suppression of Ang II-induced AF phenotypic transformation,proliferation and migration. Taken together, these results suggest a novel biologicalfunction and signaling mechanism of CREG in mouse AF regulation and that CREGdown-regulation may contribute to adventitial thickening and vascular remodeling aftervascular injury. |
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