Relationship between Perlecan and Tropoelastin Gene Expression and Cell Replication in the Developing Rat Pulmonary Vasculature

Abstract

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.

Novel Embryonic Genes Are Preferentially Expressed by Autonomously Replicating Rat Embryonic and Neointimal Smooth Muscle Cells

“…Previous data from our laboratory demonstrated that pulmonary artery and airway SMC and airway epithelial cell replication rates during development parallel those observed for aortic SMCs, 18 suggesting that an intrinsic, developmentally timed mechanism controls the replication of a variety of cell types. We examined the expression of emb8:EGAP mRNA in the developing rat lung, a tissue known to undergo extensive remodeling throughout intrauterine and early postnatal life.…”

Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic

“…However, these studies were conducted in adult models and cells and may have limited relevance to the fetal pulmonary vasculature. In the developing rat lung, maximal SMC proliferation occurred during the embryonic period, followed by a decline in the fetal period and a steady decrease after birth (5). Furthermore, the decrease in proliferation was associated with an increase in the expression of perlecan, which was predominant in nonreplicating cells.…”

Perlecan Mediates the Antiproliferative Effect of Apolipoprotein E on Smooth Muscle Cells

“…Perlecan was shown to negatively correlate with SMC proliferation (30), and it was shown to inhibit Oct-1, a growthrelated transcription factor (31). In certain cell types, however, blocking perlecan production via antisense DNA inhibited cell growth (32)(33)(34).…”