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

为探讨血小板源生长因子（PDGF）A链在高血压心血管器官过度生长中的作用，我们观察了PDGF A链反义寡核苷酸（ODN）对自发性高血压大鼠（SHR）心血管器官生长的影响。反义ODN显著抑制SHR血管平滑肌细胞（VSMC）的生长，但对正常血压的Wistar-Kyoto（WKY）大鼠的VSMC生长无明显影响。32 P反义ODN可抑制PDGF A链蛋白的生成，但不抑制PDGF A链mRNA的生成。反义寡核苷酸灌注28天后，可明显抑制血管的过度生长，并产生含或不含第6外显子的PDGF A链mRNA亚型。RT-PCR显示SHR和WKY大鼠的VSMC中存在PDGF A链的长型和短型转录本，其中长型mRNA在SHR和WKY大鼠的VSMC中更丰富 ...更多信息 循环测序分析表明，大鼠外显子6由70个核苷酸组成，与人外显子6有8个碱基不同。第6外显子反义ODN可明显抑制SHR VSMC的生长，抑制VSMC长型PDGF A链mRNA的表达，提示长型PDGF A链也参与了SHR VSMC的过度生长，探讨PDGF A链反义ODN治疗高血压心血管损害的可行性。我们在体内研究了PDGF A链反义ODN对卒中易感SHR（SHR-SP）心血管器官生长的影响。硫代磷酸连接的反义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，在主动脉和肾脏，但不影响心脏。反义ODN可明显改善SHR-SP动脉和肾组织的形态学损害，证实反义DNA抑制PDGF A链作为基因治疗手段可用于治疗高血压心血管器官损害，高血压不是高血压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