Nuthakki Vijay K, Fleser Paul S, Malinzak Lauren E, Seymour Marilyn L, Callahan Rose E, Bendick Phillip J, Zelenock Gerald B, Shanley Charles J
Department of Surgery and Research Institute, William Beaumont Hospital, Royal Oak, MI, USA.
J Vasc Surg. 2004 Jul;40(1):123-9. doi: 10.1016/j.jvs.2004.02.028.
Traditional therapies for arteriosclerotic disease often fail as a result of an exaggerated fibroproliferative response (recurrent stenosis) at the site of the intervention. Lysyl oxidase, secreted by activated vascular smooth muscle cells and fibroblasts, catalyzes a key step in the cross-linking and stabilization of collagen and elastin in the vascular wall. We hypothesized that lysyl oxidase messenger RNA (mRNA) and protein expression are time-dependent and precede collagen accumulation and luminal narrowing after arterial balloon injury in the rat.
A 2F balloon-tipped catheter was used to injure the right common carotid artery in male Sprague-Dawley rats. Injured right and control (uninjured) left common carotid arteries were harvested at 0, 0.25, 1, 3, 7, 14, 21, 28, and 60 days for mRNA quantitation and immunohistochemical analysis. Steady-state lysyl oxidase mRNA levels were quantitated with real-time reverse transcription polymerase chain reaction (TaqMan). Immunohistochemical staining with antibodies to alpha-smooth muscle cell actin and lysyl oxidase, and Movat pentachrome staining were performed for qualitative assessment of changes in the cellular and extracellular matrix components of the vessel wall. Post-injury intimal area was measured from hematoxylin and eosin-stained specimens at each time point.
When compared with sham-operated control arteries, lysyl oxidase expression in balloon-injured arteries increased significantly to 212% by day 3 after injury, and remained elevated through day 21, with a decrease toward baseline levels by day 28. Lysyl oxidase protein expression did not peak until day 14, and persisted through day 28. Collagen accumulation peaked at day 28, corresponding to the maximal increase in intimal area, with later accumulation of proteoglycans and ground substance in the intimal lesion.
Our results indicate that lysyl oxidase mRNA and protein expression is time-dependent after balloon injury of the rat carotid artery and that expression appears to precede maximal collagen accumulation and corresponding increases in intimal area. This suggests that lysyl oxidase may have an important role in stabilization of collagen and elastin at sites of vascular injury and that modulation of lysyl oxidase activity may be a viable method to prevent or reduce recurrent stenosis.
Failure of traditional therapies for ischemic arteriosclerotic disease is often due to an exaggerated fibroproliferative response (recurrent stenosis) at the site of intervention. Recurrent stenosis can be viewed as an injury-repair process, with an initial stage characterized by cellular proliferation followed by deposition of extracellular matrix. This study focuses on lysyl oxidase, a key enzyme involved in stabilization of collagen and elastin. This study demonstrates that lysyl oxidase messenger RNA and protein expression are time-dependent, preceding collagen accumulation and corresponding increases in intimal area. Accumulation of extracellular matrix is a major factor in growth of the restenotic lesion, and modulation of lysyl oxidase activity may offer a therapeutic method for decreasing or preventing recurrent stenosis.
由于在干预部位出现过度的纤维增生反应（再狭窄），动脉硬化疾病的传统治疗方法常常失败。由活化的血管平滑肌细胞和成纤维细胞分泌的赖氨酰氧化酶催化血管壁中胶原蛋白和弹性蛋白交联与稳定的关键步骤。我们推测，在大鼠动脉球囊损伤后，赖氨酰氧化酶信使核糖核酸（mRNA）和蛋白表达具有时间依赖性，且先于胶原蛋白积累和管腔狭窄出现。
使用2F球囊导管损伤雄性Sprague-Dawley大鼠的右侧颈总动脉。在0、0.25、1、3、7、14、21、28和60天采集损伤的右侧和对照（未损伤）左侧颈总动脉，用于mRNA定量和免疫组织化学分析。用实时逆转录聚合酶链反应（TaqMan）对稳态赖氨酰氧化酶mRNA水平进行定量。用抗α-平滑肌肌动蛋白和赖氨酰氧化酶抗体进行免疫组织化学染色，并用Movat五色染色法对血管壁细胞和细胞外基质成分的变化进行定性评估。在每个时间点从苏木精和伊红染色的标本测量损伤后的内膜面积。
与假手术对照动脉相比，球囊损伤动脉中的赖氨酰氧化酶表达在损伤后第3天显著增加至212%，并持续升高至第21天，到第28天降至基线水平。赖氨酰氧化酶蛋白表达直到第14天才达到峰值，并持续至第28天。胶原蛋白积累在第28天达到峰值，与内膜面积的最大增加相对应，随后在内膜病变中蛋白聚糖和基质积累。
我们的结果表明，大鼠颈动脉球囊损伤后，赖氨酰氧化酶mRNA和蛋白表达具有时间依赖性，且表达似乎先于最大胶原蛋白积累和相应的内膜面积增加。这表明赖氨酰氧化酶可能在血管损伤部位的胶原蛋白和弹性蛋白稳定中起重要作用，并且调节赖氨酰氧化酶活性可能是预防或减少再狭窄的可行方法。
缺血性动脉硬化疾病传统治疗方法的失败通常是由于干预部位出现过度的纤维增生反应（再狭窄）。再狭窄可被视为一个损伤修复过程，初始阶段以细胞增殖为特征，随后是细胞外基质的沉积。本研究聚焦于赖氨酰氧化酶，这是一种参与胶原蛋白和弹性蛋白稳定的关键酶。本研究表明，赖氨酰氧化酶信使核糖核酸和蛋白表达具有时间依赖性，先于胶原蛋白积累和相应的内膜面积增加。细胞外基质的积累是再狭窄病变生长的主要因素，调节赖氨酰氧化酶活性可能为减少或预防再狭窄提供一种治疗方法。
