Déglise Sébastien, Martin David, Probst Hervé, Saucy François, Hayoz Daniel, Waeber Gérard, Nicod Pascal, Ris Hans-Beat, Corpataux Jean-Marc, Haefliger Jacques-Antoine
Department of Thoracic and Vascular Surgery, University Hospital, Lausanne, Switzerland.
J Vasc Surg. 2005 Jun;41(6):1043-52. doi: 10.1016/j.jvs.2005.02.036.
Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity.
Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis.
Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression.
These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia.
Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.
内膜增生是血管损伤后发生的一种血管重塑过程。内膜增生所涉及的机制包括中膜平滑肌细胞的增殖、去分化以及向内膜下间隙的迁移。我们推测，协调细胞生长和分化等生理过程的缝隙连接可能参与内膜增生的发展。连接蛋白43（Cx43）的表达水平可能在内膜增生中发生改变，因此我们评估了在有或无氟伐他汀（一种3-羟基-3-甲基戊二酰辅酶A还原酶活性抑制剂）存在的情况下，培养的人隐静脉中Cx43的表达调控情况。
在旁路移植手术时获取的人隐静脉段，将其纵向打开，使管腔面朝上，并在培养中维持14天。然后对静脉片段进行组织学检查、新生内膜厚度测量、免疫细胞化学、RNA和蛋白质分析。
在四种连接蛋白（Cx37、40、43和45）中，我们重点关注Cx43和Cx40，通过实时聚合酶链反应我们发现它们在隐静脉中表达，因为它们是平滑肌细胞和内皮细胞表达的主要连接蛋白。培养14天后，组织形态计量学分析显示，在内膜增生过程中观察到内膜厚度显著增加。时间进程分析显示，Cx43逐渐上调，在培养14天后，转录水平和蛋白质水平均达到最大增加六至八倍。相比之下，在内皮细胞中大量表达的Cx40的表达未发生改变。免疫荧光显示，中膜层平滑肌细胞膜内的Cx43大量增加。在氟伐他汀存在的情况下，体外内膜增生的发展减少，且与Cx43表达降低相关。
这些数据表明，在体外内膜增生过程中Cx43增加，且氟伐他汀可阻止这种诱导，支持Cx43介导的缝隙连接通讯在人静脉内膜增生发展过程中起关键作用。
内膜增生导致的狭窄是静脉旁路移植失败的最常见原因。为了更好地理解内膜增生的发展，我们使用离体器官培养模型来研究从接受下肢旁路手术的患者获取的隐静脉。在这个模型中，血管壁的形态和功能完整性得以维持，且在培养14天后会发生显著的内膜增生。我们推测，协调细胞生长和分化等生理过程的缝隙连接可能参与内膜增生的发展。事实上，内膜增生包括平滑肌细胞增殖并迁移至内皮下间隙。细胞间通讯负责通过细胞间连接处发现的缝隙连接通道将离子和小分子从一个细胞直接转移至另一个细胞。迄今为止，尚无研究评估缝隙连接通讯是否参与人类内膜增生过程。我们通过使用上述人隐静脉内膜增生的器官培养模型对这一观点进行了研究，我们的数据支持Cx43介导的缝隙连接通讯在人静脉内膜增生发展过程中起关键作用。
