Belknap J K, Weiser-Evans M C, Grieshaber S S, Majack R A, Stenmark K R
Department of Pediatrics, University of Colorado Health Sciences Center, Denver 80262, USA.
Am J Respir Cell Mol Biol. 1999 Jan;20(1):24-34. doi: 10.1165/ajrcmb.20.1.3321.
Smooth-muscle-cell (SMC) replication and extracellular matrix protein expression are two vital and interrelated processes necessary for normal development of the vasculature. To understand better the nature of this relationship in the developing rat lung, we investigated the relationship between SMC proliferation and the expression of perlecan, a basement membrane (BM) heparan sulfate proteoglycan implicated in the control of SMC growth and differentiation, and tropoelastin (TE), a structural matrix protein not known to influence directly the replicative state of SMCs. Using bromodeoxyuridine (BrdU) incorporation to assess DNA synthesis, we first established the time course of SMC proliferation in the hilar pulmonary artery (PA) from embryonic to adult life. We found a labeling index of > 80% during the embryonic period (embryonic Day 13 [e13] to fetal Day 18 [f18]), a dramatic decline to approximately 40% during the fetal period of development, and a steady decrease in proliferation rates following birth such that, by 30 d of age, a labeling index of < 2% was noted. Using in situ hybridization, we found that although peak expression of both perlecan and TE messenger RNA (mRNA) occurred in the fetal and early postnatal periods following the major decrease in cell replication, TE mRNA expression was clearly observed in the PA as early as embryonic Day 14, whereas perlecan transcripts were virtually undetectable until fetal Day 19. Therefore, to evaluate further the relationship between cell replication and perlecan and/or TE gene expression, we used a combined in situ hybridization/BrdU immunohistochemistry technique and demonstrated that, on an individual cell basis, perlecan message was predominantly expressed by nonreplicating (BrdU-negative) PA, whereas TE mRNA was equally expressed in replicating and nonreplicating PA SMCs. Interestingly, a very similar pattern of replication and relationship to perlecan and TE mRNA expression was noted in airway SMCs and epithelial cells. Thus, in the lung as a whole, maximal expression of both the BM protein perlecan and the interstitial matrix protein TE occurs coordinately and follows the period of maximal SMC proliferation. However, in individual SMCs, perlecan mRNA expression varies inversely with DNA synthesis, whereas TE mRNA expression appears independent of the proliferative state of the cell.
平滑肌细胞（SMC）复制和细胞外基质蛋白表达是脉管系统正常发育所必需的两个重要且相互关联的过程。为了更好地理解发育中的大鼠肺中这种关系的本质，我们研究了SMC增殖与基底膜硫酸乙酰肝素蛋白聚糖（参与控制SMC生长和分化）核心蛋白聚糖的表达以及原弹性蛋白（TE，一种未知直接影响SMC复制状态的结构基质蛋白）表达之间的关系。我们使用溴脱氧尿苷（BrdU）掺入法评估DNA合成，首先确定了从胚胎期到成年期肺门肺动脉（PA）中SMC增殖的时间进程。我们发现胚胎期（胚胎第13天[e13]至胎儿第18天[f18]）标记指数>80%，在胎儿发育期间急剧下降至约40%，出生后增殖率持续下降，以至于到30日龄时，标记指数<2%。使用原位杂交，我们发现虽然核心蛋白聚糖和TE信使核糖核酸（mRNA）的峰值表达出现在细胞复制大幅减少后的胎儿期和出生后早期，但早在胚胎第14天就在PA中清楚地观察到TE mRNA表达，而核心蛋白聚糖转录本直到胎儿第19天才几乎检测不到。因此，为了进一步评估细胞复制与核心蛋白聚糖和/或TE基因表达之间的关系，我们使用了原位杂交/BrdU免疫组织化学联合技术，并证明在单个细胞水平上，核心蛋白聚糖信息主要由非复制（BrdU阴性）PA表达，而TE mRNA在复制和非复制的PA SMC中表达相当。有趣的是，在气道SMC和上皮细胞中也观察到了非常相似的复制模式以及与核心蛋白聚糖和TE mRNA表达的关系。因此，在整个肺中，基底膜蛋白核心蛋白聚糖和间质基质蛋白TE的最大表达是协调发生的，并且在SMC增殖的高峰期之后出现。然而，在单个SMC中，核心蛋白聚糖mRNA表达与DNA合成呈负相关，而TE mRNA表达似乎与细胞的增殖状态无关。
