Relationship between perlecan and tropoelastin gene expression and cell replication in the developing rat pulmonary vasculature

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.