The Effect Of Angiotensin Ⅱ Type1Receptor-associated Protein(ATRAP) Mediated By Nanoparticles On Neointion Formation In Rats

Background and ObjectiveThe morbidity of atherosclerosis in China’s urban population significantlyincreased due to the changes of lifestyle and dietary structure and other factors, thusseriously threatening people’s health. Vascular bypass surgery and percutaneousangioplasty are the main therapeutical means, but the postoperative intimalhyperplasia in vessels and anastomotic often result in a long-term unsatisfactorypatency rates, so that patients can ultimately benefit from these treatments.Gene therapy is regarded as one of the fundamental ways to solve the vasculardisorders in the future while efficient and safe vector system is one of the keyconditions for gene therapy. Nanocarriers have become the first choice for genetherapy because of their higher transfection rate, safety and low toxicity andnon-immunogenicity. Polylactic glycolic acid (PLGA), as one of the biodegradablenano-materials, has been approved by U.S. Food and Drug Administration (FDA) andsuccessfully applied in the gene transfection research in the word.Angiotensin II (Angiotinsin II, Ang II) is an important mediator of vascularpathology physiology, and an important effects of the peptide of the renin angiotensin-aldosterone system which regulates water and salt metabolism, angiotasisand activities of the sympathetic nervous system. The function of Ang II dependsnecessarily on its receptor. Type I Ang II receptor (AT1R) mainly distributes invascular smooth muscle cells (VSMC) and mediates the overwhelming majority ofthe biological effects of Ang II. So AT1R gene becomes naturally an importantcandidate gene in the research on atherosclerosis. Ang II type1receptor-relatedprotein (Angiotensin II Type I Receptor-Associated Protein, ATRAP), which was firstisolated and cloned by Dzau, VJ in laboratory at Harvard University in recent years,has already been an important promising target gene of this kind.The experiment was planned to be conducted in model rats of vascular injury andrat VSMC in vitro, while the efficiency and feasibility of gene transfection vianano-vector-mediated gene plasmid and ATRAP carrying nanoparticles wereevaluated. This study will also demonstrate the function and the cellular andmolecular mechanisms of nanoparticle-mediated ATRAP in the VSMC apoptosis andthe inhibition of intimal hyperplasia.Materials and MethodsIn this study, a copolymer of polylactic acid and polyglycolic acid (PLGA) and apolyvinyl alcohol (PVA) were used to entrap and carry the ATRAP gene plasmid andto prepare the nano-particle mixture, and then the gene plasmid is transfected into therat VSMC in vitro and the model rats of neck vascular injury via the nanoparticle-mediated gene transfection.Methods: Frist,construction of the nanoparticles-ATRAP gene transfection system:PLGA and PVA was used to entrap ATRAP gene plasmid, and the granularity and thetransfection efficiency in vivo and in vitro detected and further improved. Second, invitro study: The cytotoxic, the inhibition of VSMC proliferation and the promotion toapoptosis were observed by using VSMC transfected with nanoparticle-mediatedATRAP.Third, in vivo study: The inhibition of VSMC proliferation and the promotionto apoptosis of ATRAP were demonstrated in vivo by locally transfected nanoparticle-mediated ATRAP into rat carotid injured by a balloon catheter.Results 1.PLGA-mediated ATRAP showed good stability and no significant cytotoxicity,with a release time of about10-14days.2.The mRNA and the expressed protein products of nanoparticle-mediated ATRAPwere observed in endangium and tunica media vasorum of model rats.3.ATRAP overexpression in VSMCs inhibited Ang II-induced3H thymidinesynthesis after48h stimulation.4.ATRAP overexpression in injuried arteries significantly decreased the activationof phospho-ERK and inhibited neointimal formation.5.The inhibitory effects of ATRAP on VSMCs proliferation and neointimalformation might be due to interfering with AT1receptor-mediated activation of ERK.Conclusion1.The AT1receptor-mediated activation of ERK plays an essential role in Ang II-regulated VSMCs proliferation.2.The experiment proved that ATRAP significantly inhibited AT1R-mediatedVSMC proliferation and vascular inflammation.3.ATRAP mediated by pcDNA3vector can inhibit the proliferation of VSMC andthe intimal hyperplasia.