Development of gene therapy for the vascular proliferative diseases by antisense DNA to PDGF A-chain

为探讨PDGF a链在高血压大鼠心血管器官过度生长中的作用，我们在体外和体内研究了PDGF a链mRNA反义寡脱氧核苷酸（ODN）对自发性高血压大鼠（SHR）心血管器官生长的影响。反义ODN显著降低SHR血管平滑肌细胞（VSMC）的生长，但对正常血压Wistar-Kyoto （WKY）大鼠的细胞无明显影响。反义ODN抑制PDGF - a链蛋白的产生，但不抑制PDGF - a链mRNA的产生。体内注射32p -反义ODN导致主动脉的放射性积累大于其他器官。反义ODN输注28 d可明显减轻体内动脉的过度生长。PDGF a链mRNA的两种异构体包括或不包括外显子6。RT-PCR结果显示，SHR和WKY大鼠VSMC中PDGF a链的长链和短链转录本均存在，其中长链mRNA在SHR和WKY大鼠VSMC中更为丰富。循环测序分析表明，大鼠外显子6与人类外显子6由70个核苷酸和8个不同的碱基组成。外显子6的反义ODN显著抑制了SHR VSMC的生长，外显子6的反义ODN抑制了VSMC中PDGF长链mRNA的表达，说明PDGF长链也促进了SHR VSMC的过度生长。为了评估PDGF a链反义ODN治疗高血压心血管器官损伤的可行性，我们在体内检测了PDGF a链反义ODN对卒中易发shr （shrr - sp）患者心血管器官生长的影响。在主动脉和肾脏中，磷硫酸连接的反义ODN显著抑制生长和PDGF - A链的表达，但不影响PDGF - A链mRNA的表达，而在心脏中不受影响。反义ODN输注在形态学上明显改善了SHR-SP的动脉和肾组织损伤。由此可以证实，通过反义DNA抑制PDGF a链作为一种基因治疗，对高血压患者的心血管器官损伤是有用的，高血压并不是高血压患者心血管器官生长的主要因素。少

To evaluate the contribution of PDGF A-chain in the exaggerated growth of cardiovascular organs in hypertension, we examined the effect of antisense oligodeoxynucleotide (ODN) to PDGF A-chain mRNA on the growth of cardiovascular organs in spontaneously hypertensive rats (SHR) in vitro and in vivo. The antisense ODN significantly reduced growth of vascular smooth muscle cells (VSMC) from SHR, but not in cells from normotensive Wistar-Kyoto (WKY) rats. The antisense ODN inhibited the production of the PDGF A-chain protein, but not of the PDGF A-chain mRNA.The injection of 32P-antisense ODN in vivo led to a greater accumulation of radioactivity in the aorta than in other organs. Infusion of antisense ODN for 28 days markedly reduced the exaggerated growth of artery in vivo.Two isoforms of PDGF A-chain mRNA including or excluding exon 6 are produced. RT-PCR revealed both long- and short-form PDGF A-chain transcripts in VSMC from SHR and WKY rats, with the long-form mRNA more abundant in SH … More R VSMC.Cycle sequencing analysis revealed that rat exon 6 is composed with 70 nucleotides and 8 different bases to human exon 6. The antisense ODN to exon 6 significantly inhibited growth of SHR V.SMC.The antisense ODN to exon 6 abolished expression of the long-form PDGF A-chain mRNA in VSMC, indicating that the long-form of PDGF A-chain also contributes to the exaggerated growth of SHR VSMC.To evaluate the PDGF A-chain antisense ODN feasibility for treating the cardiovascular organ damages in hypertension, we examined the effects of the PDGF A-chain antisense ODN on cardiovascular organ growth in stroke prone-SHR (SHR-SP) in vivo. A phosphorothioate-linked antisense ODN significantly inhibited growth and PDGF A-chain expression, but did not affect expression of PDGF A-chain mRNA, in aorta and kidney but not in heart. Infusion of the antisense ODN considerably improved the arterial and renal tissue damage in SHR-SP morphologically.From these findings, it can be confirmed that suppression of PDGF A-chain by the antisense DNA is useful for treating the cardiovascular organ damages in hypertension as a gene therapy, and that high blood pressure is not the primary factor responsible for cardiovascular organ growth in hypertension. Less