Chen Y M, Wu K D, Tsai T J, Hsieh B S
Department of Medicine, National Taiwan University Hospital, Taipei, Taiwan.
J Mol Cell Cardiol. 1999 Apr;31(4):773-83. doi: 10.1006/jmcc.1998.0910.
There is growing evidence that pentoxifylline (PTX) may have potential value as an antiproliferative and antifibrogenic agent. To assess whether this drug may be of use in the prevention of atherosclerosis or restenosis after angioplasty, we investigated the ability of PTX to inhibit proliferation and collagen synthesis in rat vascular smooth muscle cells (VSMCs) under both basal and platelet-derived growth factor (PDGF)- or transforming growth factor-beta (TGF-beta)- stimulated conditions. Intracellular cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were measured in confluent cells using enzyme immunoassay kits. Cell proliferation was measured by methyltetrazolium assay. Cell cycle distribution was determined by flow cytometry. Total collagen synthesis was measured by 3H-proline incorporation assay. Expression of collagen alpha 1(I) and collagen alpha 1(III) mRNAs was detected by northern blotting. Addition of PTX to VSMC cultures suppressed both basal and PDGF-AB (25 ng/ml)-driven cell proliferation, in conjunction with a cell cycle blockade at the G1/S phase at 24 h. This effect was predominantly cAMP-dependent, as PTX increased cAMP in a dose-dependent manner (0.03 to 0.33 mg/ml) but not cGMP level, and the addition of dibutyryl-cAMP (0.2 to 2 m m) closely mimicked the effect of PTX. Furthermore, co-incubation with a selective inhibitor of cAMP-dependent protein kinase (PKA), H-89 (2.0 microm), or an N -myristoylated PKA pseudosubstrate nonapeptide, m-phi PKA (10 microm), prevented the antimitogenic effect of PTX. PTX also suppressed both basal and TGF- beta 1-augmented collagen alpha 1(I) and collagen alpha 1(III) mRNA levels beginning at 24 h, and attenuated both basal and TGF-beta 1 (5 ng/ml)-stimulated total collagen synthesis at 48 h. Co-incubation with H-89 or m-phi PKA reversed PTX-attenuated collagen alpha 1(I) and collagen alpha 1(III) mRNA levels at 24 h. These data suggest that the antimotigenic and anticollagen effects of PTX were mediated predominantly through a cAMP-PKA effector pathway. The dual effect of PTX on VSMC proliferation and collagen synthesis may form the rationale for animal or clinical trials for the treatment of vascular occlusion due to atherosclerosis and restenosis following angioplasty.
越来越多的证据表明，己酮可可碱（PTX）作为一种抗增殖和抗纤维化药物可能具有潜在价值。为了评估这种药物是否可用于预防动脉粥样硬化或血管成形术后再狭窄，我们研究了PTX在基础条件以及血小板衍生生长因子（PDGF）或转化生长因子-β（TGF-β）刺激条件下抑制大鼠血管平滑肌细胞（VSMC）增殖和胶原合成的能力。使用酶免疫分析试剂盒测量汇合细胞中的细胞内环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）水平。通过甲基四氮唑法测量细胞增殖。通过流式细胞术确定细胞周期分布。通过3H-脯氨酸掺入法测量总胶原合成。通过Northern印迹检测胶原α1（I）和胶原α1（III）mRNA的表达。向VSMC培养物中添加PTX可抑制基础和PDGF-AB（25 ng/ml）驱动的细胞增殖，并在24小时时导致细胞周期在G1/S期阻滞。这种作用主要依赖于cAMP，因为PTX以剂量依赖性方式（0.03至0.33 mg/ml）增加cAMP，但不增加cGMP水平，并且添加二丁酰-cAMP（0.2至2 mM）紧密模拟PTX的作用。此外，与cAMP依赖性蛋白激酶（PKA）的选择性抑制剂H-89（2.0 microm）或N-肉豆蔻酰化的PKA假底物九肽m-phi PKA（10 microm）共同孵育可阻止PTX的抗有丝分裂作用。PTX还从24小时开始抑制基础和TGF-β1增强的胶原α1（I）和胶原α1（III）mRNA水平，并在48小时时减弱基础和TGF-β1（5 ng/ml）刺激的总胶原合成。与H-89或m-phi PKA共同孵育可逆转PTX在24小时时减弱的胶原α1（I）和胶原α1（III）mRNA水平。这些数据表明，PTX的抗有丝分裂和抗胶原作用主要通过cAMP-PKA效应途径介导。PTX对VSMC增殖和胶原合成的双重作用可能为治疗动脉粥样硬化和血管成形术后再狭窄引起的血管闭塞的动物或临床试验提供理论依据。
