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

为探讨PDGF A链在高血压心血管器官过度生长中的作用，我们在体内外观察了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。反义寡核苷酸显著抑制自发性高血压大鼠血管平滑肌细胞(VSMC)的生长，但对正常血压Wistar-京都(WKY)大鼠的VSMC生长无明显影响。反义ODN可抑制PDGF A链蛋白的生成，但不抑制PDGF A链mRNAs的生成。体内注射32P反义ODN后，放射性在主动脉中的蓄积明显高于其他器官。体内注射反义ODN 28d后，动脉的过度生长明显减少，可产生包括或不包括外显子6在内的两种不同亚型的PDGF A链mRNA。RT-PCR法检测到自发性高血压大鼠和WKY大鼠血管内皮细胞均有长型和短型PDGFA链转录产物，其中SH-…的长型表达更为丰富大鼠外显子6由70个核苷酸和人类外显子6的8个不同碱基组成。外显子6的反义ODN显著抑制SHR VSMC的生长。外显子6的反义ODN抑制了VSMC中长型PDGF A链mRNA的表达，提示PDGF A链的长型也是SHR VSMC过度生长的原因之一。我们研究了PDGF A链反义寡核苷酸对卒中易感自发性高血压大鼠(SHR-SP)心血管器官生长的影响。硫代修饰的反义寡核苷酸可显著抑制大鼠的生长和PDGF A链的表达，但不影响PDGF A链mRNA在主动脉和肾脏的表达，但不影响心脏的表达。结果表明，反义DNA抑制PDGF A链可作为基因治疗高血压心血管器官损害的有效手段，而高血压不是高血压心血管器官生长的主要因素。较少

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