Department of Chemical and Biochemical Engineering, Faculty of Engineering, The University of Western Ontario, London, Ontario, Canada.
Biomaterials. 2012 Oct;33(29):7047-56. doi: 10.1016/j.biomaterials.2012.06.044. Epub 2012 Jul 13.
Incorporation of endogenous elastin, a key structural component of the vascular extracellular matrix (ECM), is an important requirement for engineered vascular tissues. In addition to providing elastic recoil of the tissue, elastin influences cell function and promotes cell signaling by interacting with specific cell surface receptors. Although progress has been made in understanding the mechanisms of in vivo elastin expression and incorporation into fibers, it is notably absent from engineered vessels. Recently we showed that the three-dimensional (3D) scaffold topography was able to upregulate elastin synthesis by human coronary artery smooth muscle cells (HCASMC). The present study was undertaken to explore the molecular mechanisms responsible for 3D scaffold-induced elastin gene transcription. Here, we show several lines of evidence that signal transduction pathway leading to elastin gene expression by HCASMC cultured in synthetic 3D scaffolds to be strikingly different from two-dimensional (2D) surfaces. In 3D scaffolds, α5β1 integrin engagement by HCASMC was significantly reduced and the putative focal adhesion kinase (FAK) was poorly phosphorylated concomitant with FAK and protein tyrosine kinase Pyk2 downregulation. FAK-associated adhesion proteins vinculin and paxillin were also significantly downregulated by the 3D scaffold topography. Furthermore, contrary to 2D cultures, HCASMC cultured on 3D scaffolds had no Rho activation suggesting pliability of the elastomeric synthetic scaffold. Elastin expression in 3D cultures followed Ras-ERK1/2 signal transduction pathway and was further dependent on endogenously expressed interleukin-1β (IL-1β). Blocking of ERK1/2 activation using a pharmacologic inhibitor reduced both elastin and IL-1β gene expressions in 3D cultures. Transient transfection of IL-1β using siRNA, however, did not affect ERK1/2 activation but downregulated elastin gene expression suggesting that endogenous IL-1β acts downstream from ERK1/2. Taken together, results of the present study provide evidence that endogenous IL-1β play a role in elastin gene upregulation and, that this upregulation is mediated by the Ras-ERK1/2 pathway in 3D cultures.
内源性弹性蛋白是血管细胞外基质（ECM）的关键结构成分，它的掺入是工程化血管组织的一个重要要求。弹性蛋白除了提供组织的弹性回弹外，还通过与特定的细胞表面受体相互作用来影响细胞功能并促进细胞信号转导。尽管人们在理解体内弹性蛋白表达和掺入纤维的机制方面已经取得了进展，但在工程化血管中却明显缺乏弹性蛋白。最近，我们发现三维（3D）支架拓扑结构能够上调人冠状动脉平滑肌细胞（HCASMC）的弹性蛋白合成。本研究旨在探讨负责 3D 支架诱导弹性蛋白基因转录的分子机制。在这里，我们提供了几条证据表明，与在二维（2D）表面培养的 HCASMC 相比，培养在合成 3D 支架中的 HCASMC 的信号转导途径导致弹性蛋白基因表达的方式有很大的不同。在 3D 支架中，HCASMC 中α5β1 整合素的结合显著减少，假定的粘着斑激酶（FAK）的磷酸化程度较差，同时 FAK 和蛋白酪氨酸激酶 Pyk2 的下调。FAK 相关的黏附蛋白 vinculin 和 paxillin 也被 3D 支架拓扑结构显著下调。此外，与 2D 培养物相反，在 3D 支架上培养的 HCASMC 没有 Rho 激活，这表明弹性体合成支架具有柔韧性。3D 培养物中的弹性蛋白表达遵循 Ras-ERK1/2 信号转导途径，并且进一步依赖于内源性表达的白细胞介素-1β（IL-1β）。使用药理学抑制剂阻断 ERK1/2 激活会降低 3D 培养物中的弹性蛋白和 IL-1β 基因表达。然而，使用 siRNA 瞬时转染 IL-1β 并不影响 ERK1/2 的激活，但下调了弹性蛋白基因的表达，这表明内源性 IL-1β 作用于 ERK1/2 的下游。总之，本研究的结果提供了证据表明内源性 IL-1β 在弹性蛋白基因的上调中起作用，并且这种上调是由 3D 培养物中的 Ras-ERK1/2 途径介导的。
