Mechanisms of Airway Smooth Muscle Hypertrophy

描述(由申请人提供)：早产儿存活率增加的同时，支气管肺发育不良(BPD)的发生率也增加。在“新的BPD”中，肺泡间隔由胶原和1-平滑肌肌动蛋白阳性、转化生长因子(TGF)阳性的肌成纤维细胞增厚。我们从早产儿的气管抽吸物中分离出间充质基质细胞。这些细胞产生转化生长因子β1，并接受转化生长因子β诱导的肌成纤维细胞分化，这表明在没有其他信号的情况下，肌成纤维细胞分化是间充质基质细胞特化的默认程序。间充质基质细胞似乎也是BPD发生的有效生物标志物；考虑到性别、出生体重和胎龄的潜在影响，分离间充质基质细胞使调整后的BPD的优势比增加了31倍以上。最后，我们的微阵列数据显示，基质细胞表达高水平的编码基质蛋白的mRNAs，基质蛋白是调节细胞-基质相互作用和纤维沉积的非结构性细胞外基质蛋白的一个亚家族。综上所述，这些数据支持我们的普遍假设，即间充质基质细胞肌成纤维细胞分化和基质细胞蛋白表达在BPD的发病机制中起关键作用。为了验证这一假设，我们提出了以下具体目标：1.新生儿肺间充质基质细胞基质细胞基质蛋白表达与临床结局的相关性。我们假设：(I)间充质基质细胞维持稳定的促纤维化表型的基质细胞蛋白表达与肌成纤维细胞分化相结合；(Ii)基质细胞蛋白结缔组织生长因子(CTGF)、SPARC和Periostin的表达预测BPD的发生。2.检测基质细胞蛋白表达对新生大鼠肺间充质基质细胞增殖分化的影响，以及糖原合成酶-3β和连环蛋白在肌成纤维细胞分化中的作用。我们假设(I)基质细胞蛋白诱导间充质基质细胞增殖，并且是转化生长因子β诱导的肌成纤维细胞分化所必需的；(Ii)肌成纤维细胞的分化依赖于NOX4/GSK-3？/？连环蛋白信号。3.体内检测基质细胞蛋白及其下游信号中间体对BPD表型的表达、定位、需求和充分性。我们假设：(I)成纤维细胞特异性CTGF的表达足以满足BPD的表型；(Ii)SPARC和Periostin是高氧诱导的肺泡减少和间质纤维化所必需的；iii)间充质特异性抑制GSK-3足以满足BPD的表型；以及iv)BPD婴儿肺中基质细胞蛋白的表达增加。这项工作的完成将为BPD的发病机制提供深入的认识，导致更准确的生物标志物的开发，并为基质细胞蛋白在肺纤维化中的作用提供新的信息。

DESCRIPTION (provided by applicant): Increased survival of very premature infants has been accompanied by an increased incidence of bronchopulmonary dysplasia (BPD). In the "new BPD," alveolar septa are thickened with collagen and 1-smooth muscle actin-positive, transforming growth factor (TGF)-¿-positive myofibroblasts. We have isolated mesenchymal stromal cells from the tracheal aspirates of premature infants. These cells produce TGF-¿1 and undergo TGF-¿-induced myofibroblastic differentiation, suggesting that, in the absence of other signals, myofibroblastic differentiation represents the "default program" for mesenchymal stromal cell specialization. Mesenchymal stromal cells also appear to be potent biomarkers for the development of BPD; accounting for the potential influences of gender, birth weight and gestational age, isolation of mesenchymal stromal cells increased the adjusted odds ratio of BPD over 31-fold. Finally, our microarray data show that stromal cells express high levels of mRNAs encoding matricellular proteins, a subfamily of non-structural extracellular matrix proteins that regulate cell-matrix interactions and fiber deposition. Together, these data support our general hypothesis that mesenchymal stromal cell myofibroblastic differentiation and matricellular protein expression play critical roles in BPD pathogenesis. To test this hypothesis, we propose the following Specific Aims: 1. Correlate neonatal lung mesenchymal stromal cell matricellular protein expression with clinical outcomes. We hypothesize that: i) mesenchymal stromal cells maintain a stable pro-fibrotic phenotype of matricellular protein expression that is coupled with myofibroblastic differentiation; and (ii) expression of the matricellular proteins connective tissue growth factor (CTGF), SPARC and periostin predicts BPD development. 2. Examine the effects of matricellular protein expression on neonatal lung mesenchymal stromal cell proliferation and differentiation, as well as the roles of glycogen synthase kinase (GSK)-3¿ and ¿-catenin in myofibroblastic differentiation in vitro. We hypothesize that (i) matricellular proteins induce mesenchymal stromal cell proliferation and are required for maximal TGF-¿-induced myofibroblastic differentiation; and (ii) myofibroblastic differentiation is dependent on Nox4/GSK-3¿/¿-catenin signaling. 3. Examine the expression, localization, requirement and sufficiency of matricellular proteins and their downstream signaling intermediates for the BPD phenotype in vivo. We hypothesize that: i) fibroblast-specific expression of CTGF is sufficient for the BPD phenotype; (ii) SPARC and periostin are required for hyperoxia-induced hypoalveolarization and interstitial fibrosis; iii) mesenchyme-specific inhibition of GSK-3¿ is sufficient for the BPD phenotype; and iv) matricellular protein expression in increased in the lungs of infants with BPD. Completion of this work will provide insight into the pathogenesis of BPD, lead to the development of more accurate biomarkers, and provide new information on the role of matricellular proteins in lung fibrosis.