Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves
Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves
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DOI：
10.1152/ajpheart.01305.2007
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发表时间：
2008-04-01
影响因子：
4.8
通讯作者：
McCulloch, C. A.
中科院分区：
文献类型：
--
作者：
Pho, M.;Lee, W.;McCulloch, C. A.
瓣膜间质细胞（VIC）群体中肌成纤维细胞的形成有助于纤维化性瓣膜疾病。我们检测了猪主动脉瓣VICs的肌成纤维细胞分化。在正常瓣膜中，对α -平滑肌肌动蛋白（α - sma，一种肌成纤维细胞标记物）免疫染色的细胞很少（0.69 +/- 0.48%），但在喂食致动脉粥样硬化饮食的动物的硬化瓣膜中，肌成纤维细胞在空间上聚集且丰富（31.2 +/- 6.3%）。在从正常瓣膜培养的VIC群体中，sma阳性的肌成纤维细胞也在空间上聚集，数量丰富（1代后阳性细胞21%），并染色为I型胶原和vimentin，但未染色为desmin。对于干细胞的分析，用Hoechst 33342染色的分离细胞的双色流式细胞术显示0.5%的vic是侧群细胞；无SMA染色。经过培养，分选的侧群体细胞产生了85%的sma阳性细胞，这表明一些肌成纤维细胞来自具有干细胞特征的罕见群体。在刚性胶原基质上培养的细胞在培养5天后能够形成肌成纤维细胞，而在柔性胶原基质上培养的细胞则完全阻断了肌成纤维细胞的形成。外源拉伸力也显著增加了VICs中SMA的表达。使用同位素编码亲和标签和质谱技术鉴定vic肌成纤维细胞分化过程中的差异表达蛋白。在鉴定的9种蛋白中，cofilin和磷酸化cofilin的表达在有利于肌成纤维细胞分化的条件下强烈增加。用小干扰RNA敲低cofilin抑制胶原凝胶收缩并减少肌成纤维细胞分化，这是通过SMA并入应力纤维来评估的。与正常瓣膜相比，病变瓣膜在聚集细胞中显示出与SMA共定位的cofilin的免疫染色。我们得出结论，在vic中，cofilin是体内和体外肌成纤维细胞的标记物，这些细胞来自罕见的干细胞群体，需要刚性基质才能形成。
The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for alpha-smooth muscle actin (alpha-SMA, a myofibroblast marker) were rare (0.69 +/- 0.48%), but in sclerotic valves of animals fed an atherogenic diet, myofibroblasts were spatially clustered and abundant (31.2 +/- 6.3%). In cultured VIC populations from normal valves, SMA-positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage), and stained for collagen type I and vimentin but not desmin. For an analysis of stem cells, two-color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated similar to 85% SMA-positive cells, indicating that some myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Exogenous tensile force also significantly increased SMA expression in VICs. Isotope-coded affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the nine proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with small-interfering RNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by the SMA incorporation into stress fibers. When compared with normal valves, diseased valves showed strong immunostaining for cofilin that colocalized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.