Nuclear Factor--κB's Role In Proliferation Of Vascular Smooth Muscle Cell Induced By Thrombin And The Intervention Of Its Anti-sense RNA

Background and Purpose: Such cardiovascular diseases as atherosclerosis (AS) andrenarrowing after percutaneous coronary intervention (PCI) are all pathologically basedon the proliferation of vascular smooth muscle cells (VSMCs) , which is connectedwith the participation of growth factor and such vascular bioactive peptides as thrombin.Pure inhibition of some specific factor cannot totally prevent focus from forming, so ithas become the research orientation towards effectively preventing the vascularproliferation-associated diseases to regulate the key gene of such final common paths ascellular proliferation, differentiation and migration. As a key factor serving to regulatein a number of ways including genetic transcription, nuclear factor--κB(NF-κB)regulates the expression of several kinds of target genes as cytokines, growth factors,adhesion molecules and chemokines and closely concerns such cardiovascular diseasesas the AS attack, hypertension, and acute coronary syndrome. It is already known thatNF-κB path also exists in VSMCs and is also the inevitable way to activate for SMCproliferation, so regulating the activity of NF-ΚB is an ideal therapeutic target. Thedurability of existing drug treatment is affected by its own unavoidable side-effect andtrivial application, so genetic treatment has become the development trend of futurecure by virtue of such characteristics as phisiological purity, more obvious targetability,lighter side-effect of its preparation, and the long-term effect upon one administration.As a kind of serine protease and mitogen, thrombin not only plays an important role informing thrombus in vascular walls of damaged arteries, but also can regulate VSMCsproliferation through a number of links. Lots of previous researches all stressedblocking the function of thrombin by inhibiting the expression of thrombin receptorgene so as to inhibit VSMCs proliferation. However, so far no reports have been foundthat deal with the relations between VSMCs proliferation induced by thromin and NF-κB path in VSMC. Given the said situation, this research attempts to probe into thedose-effect and time-effect relations of thrombin promotion of VSMCs proliferation,the relations between its guidance and NF-κB path and possible mechanism concernedby using thrombin as VSMC mitogen. It also tries to construct adenovirus vectorexpressing NF--κB anti-sense RNA while testing the changes in the expression of NF-κB gene in infected VSMC, probes into how the pre-treatment of recombinantadenovirus expressing NF-κB anti-sense RNA acts on VSMCs proliferation inducedby thrombin in vitro.Method: Use generation 3-5 monocell suspension of VSMCs for the experiment.0.01, 0.1, 0.2, 0.5, 1, 2U/ml thrombin stimulates VSMCs 24h and 0.5U/ml thrombinVSMCs 1,2,6,12,24,48h; determine the proliferation rate of VSMCs cells with WST-1metabolic activity and 3 H-TdR incorporation rate; after 0.5U/ml thrombin hasstimulated VSMCs for the predetermined time, test the orientation of NF-κB inVSMCs and the protein expression of NF-κB in nucleus of VSMCs withimmunefluorescent microscope technique and western blot in basic condition and withthrombin interference; after pre-treating 100umol/L PDTC (NF-κB special inhibitor),investigate the proliferation rate induced by thrombin; after pre-treating 10umol/L DPI(NAD(P)H oxidase inhibitor), determine the reactive oxygen with the ultravioletspectroscopic photometer. Total RNA was extracted from VSMCs and used to amplifythe 1653bp fragment of NF-κB (p65) gene by RT-PCR. The amplified product wasinserted into the pMD18-T vector and identified subsequently by enzyme digestionanalysis and sequencing. The downstream 269bp of NF-κB (p65) gene was amplifiedfrom the recombinant pMD18-T/1653 plasmid and cloned in reverse orientation intothe eukaryotic expression vector, pcDNA3.1(+), under the CMV promoter. The intactantisense RNA expression frame from pcDNA3.1(+)/269 was inserted intoentryvector pENTR4 to form medium recombinant pENTR4/CMV/269 plasmid, followedby homologous recombination technique, the expression frame was integrated intoadenovirus. The linearized recombinant adenovirus plasmid, pAd/CMV/269, infectedthe monolayer 293 cells, the adenovirus packaging cell line. Real-time PCR andwestern blot were employed to determine the changes of NF-κB (p65) gene expressionlevel in the virus-infected VSMCs. After pre-treating the recombinant adenovirusexpressing NF-κB anti-sense RNA in VSMCs cultured, determine the proliferationrate of VSMCs induced by 0.5U/ml thrombin in the said way.Result:Part I: Thrombin with concentration of 0.2～2U/ml apparently promotes VSMCsproliferation, which differs irremarkably from the promotion of VSMCs proliferationfor thrombin with concentration of 0.5～2U/ml at 24h (P>0.05). The proliferation curvefor 0.5U/ml thrombin exhibits the change in the shape of double peaks against thecontrast group. The peak values at 1h and 24h show that thrombin promotion ofVSMCs proliferation is not only rapid and brief but also delayed and retarding. NF-κBof VSMCs in basic condition is mainly distributed in cytoplasm while that of VSMCsinterfered by thrombin in nucleus with NF-κB obviously reduced in cytoplasm. ThePDTC pre-treatment can obviously inhibit the thrombin promotion of VSMCsproliferation (P<0.01). DPI pre-treatment can obviously inhibit the reactive oxygenfrom being induced by thrombin (P<0.01), which shows that thrombin may activateNF-κB path through oxidative stress.Part Ⅱ: A 1653bp fragment of NF-κB (p65) gene was amplified from VSMCs, itssequence analysis was documented as expected. The transition-vectors contained269bp of reverse sequence were identified by restrictive endonuclease and PCRanalysis. Recombinant adenovirus that expressed NF-κB (p65) antisense RNA wasconstructed correctly and the titer of virus was generally up to 9.23×109 plaque formunits per milliliter (pfu/ml). In vitro, real-time PCR and western blot results showedthat the NF-κB (p65) mRNA and protein expression level in the infected VSMCs wasmarkedly reduced in compare with normal cells.Part III: The pre-treatment of recombinant adenovirus expressing NF--κB anti-senseRNA can obviously inhibit thrombin guidance of VSMCs proliferation (P<0.01) at therate of >30%, which is similar to the inhibition of VSMCs proliferation by NF-κBspecial inhibitor PDTC with no statistical difference in between (P>0.05).Conclusion:Part I: Thrombin with concentration of 0.2～0.5 U/ml depends a lot on concentrationin promoting VSMCs proliferation. The proliferation curve guided by thrombin exhibitsthe change in the shape of double peaks in terms of time to suggest that thrombinpromotion is delayed. Thrombin can activate NF-κB of VSMCs to make it transferfrom cytoplasm to nucleus. VSMCs proliferation induced by thrombin is connectedwith NF-κB path, so NF-κB may be the common path for the signal transferenceinside the VSMCs proliferated.Part II: On the basis of DNA recombination technology, the gateway cloning system ofGateway TM is adopted to successfully construct the adenovirus as expression vectorof NF-κB anti-sense RNA and obtain virus supernatants which have higher virus titer.After being infected by recombinant adenovirus expressing NF-κB anti-sense RNA,target cells—rat VSMCs express NF-κB anti-sense RNA to reduce the NF-κB (p65)mRNA and protein expression level inside.Part III: After being infected by recombinant adenovirus expressing NF-κBanti-sense RNA, VSMCs express NF-κB anti-sense RNA to effectively inhibit ofVSMCs proliferation induced by thrombin. In the same conditions, NF-κB anti-senseRNA and NF-κB special inhibitor play the same role in inhibiting VSMC proliferationat the rate of over 30%, which can be applied to successive basic research and clinicalinterference treatment.