Abstract
We wish to understand the process of smooth muscle cell (SMC) proliferation and maturation during late fetal development and have examined some of the molecular changes associated with blood vessel maturation in late gestational and early neonatal life. By differential screening of a fetal aortic smooth muscle cDNA library, we identified a gene (F-31) that was developmentally regulated in aortic smooth muscle. The F-31 gene encodes a 23-kb RNA that was highly expressed in fetal aortic smooth muscle (25-day gestation), was lower in newborns, and was undetectable in the aortic smooth muscle of 4-week-old animals. F-31 was also highly expressed in fetal muscle, esophagus, heart, liver, lung, and placenta; its expression was lower in skin, kidney, and brain. By contrast, the expression of F-31 was low or undetectable in the corresponding tissue of adult animals. DNA sequence analysis of cDNAs encoding F-31 and data base comparison revealed a 73% homology with a previously identified, developmentally regulated gene called H19. We also found that insulin-like growth factor II (IGF-II) expression was developmentally regulated in smooth muscle. However, unlike F-31, expression of IGF-II was undetectable in the aortic smooth muscle of newborn animals. Analysis of the mRNA level of several genes that encode cytoskeletal proteins in neonatal, newborn, and adult smooth muscle indicates that total actin mRNA level, a-smooth muscle actin, and a-tropomyosin mRNA levels were similar between the late gestational period and 4 weeks after birth. By contrast, the mRNA levels of smooth myosin heavy chain increased sixfold. Expression of both F-31 and IGF-II was maintained in cultured cells obtained from fetal smooth muscle but was not detectable in adult SMCs. Interestingly, their expression was shut off in high-density growth-arrested cells. However, growth-arrested cells treated with 0.5% serum, 10-6 M insulin, and 5 ,g/ml transferrin reexpressed F-31 but not IGF-II. Morphological comparison of fetal and adult SMCs in culture revealed that although proliferating fetal and adult SMCs were indistinguishable, in high density cultures, fetal cells fail to form the "hill and valley" morphology characteristic of adult SMCs. (Circulation Research 1992;71:711-719) KEY WoRDs * smooth muscle cells * development * gene expression * regulation V ascular smooth muscle cell (SMC) migration, proliferation, and differentiation are an integral aspect of blood vessel development, yet a relatively small amount of information is available concerning the molecular events associated with their development.1-4 It is known that during fetal life SMCs proliferate rapidly and are fibroblastic in morphology but that their replication rate decreases soon after birth.56 Associated with this decrease in DNA synthesis is an increase in cells with myofilament bundles, well-From the Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Md.
Abstract
The nucleotide sequence reported in this paper has been submitted to the GenBank'/EMBL Data Bank with accession number M97348.
Identification and Characterization of Developmentally Regulated Genes in Vascular Smooth Muscle Cells
David K.M. Han 
and Gene Liau
We wish to understand the process of smooth muscle cell (SMC) proliferation and maturation during late fetal development and have examined some of the molecular changes associated with blood vessel maturation in late gestational and early neonatal life. By differential screening of a fetal aortic smooth muscle cDNA library, we identified a gene (F-31) that was developmentally regulated in aortic smooth muscle. The F-31 gene encodes a 23-kb RNA that was highly expressed in fetal aortic smooth muscle (25-day gestation) , was lower in newborns, and was undetectable in the aortic smooth muscle of 4-week-old animals. F-31 was also highly expressed in fetal muscle, esophagus, heart, liver, lung, and placenta; its expression was lower in skin, kidney, and brain. By contrast, the expression of F-31 was low or undetectable in the corresponding tissue of adult animals. DNA sequence analysis of cDNAs encoding F-31 and data base comparison revealed a 73% homology with a previously identified, developmentally regulated gene called H19. We also found that insulin-like growth factor II (IGF-II) expression was developmentally regulated in smooth muscle. However, unlike F-31, expression of IGF-II was undetectable in the aortic smooth muscle of newborn animals. Analysis of the mRNA level of several genes that encode cytoskeletal proteins in neonatal, newborn, and adult smooth muscle indicates that total actin mRNA level, a-smooth muscle actin, and a-tropomyosin mRNA levels were similar between the late gestational period and 4 weeks after birth. By contrast, the mRNA levels of smooth myosin heavy chain increased sixfold. Expression of both F-31 and IGF-II was maintained in cultured cells obtained from fetal smooth muscle but was not detectable in adult SMCs. Interestingly, their expression was shut off in high-density growth-arrested cells. However, growth-arrested cells treated with 0.5% serum, 10-6 M insulin, and 5 ,g/ml transferrin reexpressed F-31 but not IGF-II. Morphological comparison of fetal and adult SMCs in culture revealed that although proliferating fetal and adult SMCs were indistinguishable, in high density cultures, fetal cells fail to form the "hill and valley" morphology characteristic of adult SMCs. (Circulation Research 1992;71:711-719) KEY WoRDs * smooth muscle cells * development * gene expression * regulation V ascular smooth muscle cell (SMC) migration, proliferation, and differentiation are an integral aspect of blood vessel development, yet a relatively small amount of information is available concerning the molecular events associated with their development.1-4 It is known that during fetal life SMCs proliferate rapidly and are fibroblastic in morphology but that their replication rate decreases soon after birth.56 Associated with this decrease in DNA synthesis is an increase in cells with myofilament bundles, well-From the Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Md.
and Gene Liau
The nucleotide sequence reported in this paper has been submitted to the GenBank'/EMBL Data Bank with accession number M97348.
and Gene Liau
Supported in part by National Institutes of Health grant HL-37510. G.L. is a recipient of Research Career Development Award HL-02449. D.H. performed this work in partial fulfillment of the requirements for a Doctor of Philosophy degree from the Department of Genetics, George Washington University, Graduate defined basal laminae, and little or no biosynthetic organelles.2,57 Analysis of cytoskeletal and contractile protein contents in SMCs during development indicates that a-smooth muscle actin, desmin, myosin heavy chain isoform 2, high molecular weight caldesmon, and metavinculin all increase as SMCs begin to acquire the contractile phenotype.5'8-12 By contrast, the expression of low molecular weight caldesmon and an embryonic myosin heavy chain isoform decrease dramatically during this process.1-13 These changes provide important clues toward understanding the developmental maturation of vascular SMCs. Interestingly, cultured SMCs obtained from the blood vessel of newborn rats express elevated levels of both platelet-derived growth factor B chain and cytochrome P4501A1 when compared with adult SMCs, and these newborn cells have a discrete epithelioid morphology. [14] [15] [16] These results suggest that distinct developmentally affiliated SMC phenotypes can be maintained in culture.
and Gene Liau
The in vivo morphological and biochemical differences observed between proliferating fetal SMCs and quiescent adult medial SMCs are very similar to those observed between proliferating cells in vascular lesions and normal adult SMCs.3,12,13,15,16 These similarities have led to the suggestion that proliferating SMCs in atherosclerosis and hypertension may recapitulate earlier developmental events. 3, 4 It is possible that elucidation of the developmental pathway of SMCs will provide important insights toward understanding aberrant SMC proliferation in the adult.
and Gene Liau
We reasoned that the identification of additional genes that are expressed in fetal but not adult smooth muscle may provide mechanistic insights toward understanding SMC development. Toward this end, we have used differential cDNA cloning procedures to attempt to identify genes that are expressed in fetal aortic smooth muscle but not in the adult counterpart. To avoid the isolation of genes that are growth associated but not developmentally regulated, we selected cDNAs that are expressed in fetal smooth muscle and not expressed in adult smooth muscle or proliferating cultured adult SMCs. In the present study, we report the isolation and characterization of a developmentally regulated gene in rabbit smooth muscle (F-31). In addition, we also identified insulin-like growth factor II (IGF-II) as a fetal-specific gene in aortic SMCs. Developmental regulation of F-31 and IGF-II was maintained in vitro and was modified by the growth state of cultured fetal SMCs.
Materials and Methods Materials
Materials used were as follows: fetal bovine serum (FBS, Hyclone Laboratories Inc., Logan, Utah), cDNA synthesis and random prime-labeling reagents (Boehringer Mannheim Corp., Indianapolis, Ind.), Agt10 and A Zap II cDNA cloning vectors and packaging reagents (Stratagene Inc., La Jolla, Calif.), DNA-sequencing reagents ( 
Cell Culture
Vascular SMCs were isolated from the thoracic aortas of fetal, newborn, and 4-week-old New Zealand White rabbits by enzymatic digestion as previously described.21 SMCs were cultured in medium 199 supplemented with 10% FBS, 4 mM L-glutamine, 100 units/ml penicillin G sodium, 100 jug/ml streptomycin sulfate, and 0.25 gg/ml amphotericin B. SMCs were positive for a-smooth muscle actin as determined by immunofluorescence using a monoclonal antibody that specifically recognizes a-smooth muscle actin.17 Cells were routinely passaged at a 1:5 ratio and used between passages 4 and 6.
Cell Culture
Preconfluent cells were harvested at 60-75% confluence. Postconfluent cells were harvested 5-7 days after SMCs had reached confluence. For some experiments, postconfluent cells were fed with medium containing 0.5% FBS, 10`6 M insulin, and 5 ,ug/ml transferrin for 3 days before harvest.
RNA Isolation and Preparation of cDNA Library
Fetal rabbit aortas were stripped of adventitia under a large magnifying glass. The aorta was then cut open, and the endothelial layer was scraped off. The remaining medial SMCs were homogenized in guanidine isothiocyanate, and total RNA was isolated by centrifugation through a cesium chloride solution. Poly(A+) mRNA was purified by passing total RNA through an oligo (dT)-cellulose column. cDNA synthesis was carried out using avian myeloblastosis virus reverse transcriptase and RNase H/DNA polymerase 1,22 and the cDNA was subsequently cloned into a AgtlO vector using EcoRI linkers as previously described. 23 This library contained 1.1x106 individual plaques with an average cDNA insert size of 800 bp. Twenty thousand individual plaques were screened by differential hybridization using 32P-labeled cDNA generated from fetal rabbit aorta mRNA and proliferating adult cultured SMC mRNA by a previously described method. 23 The 450-bp cDNA (F-31) was used to screen a rabbit placenta A Zap II cDNA library synthesized using random primers. cDNA clones of 1.2 kb and 1.8 kb were isolated and characterized by DNA sequencing.
RNA Analysis and DNA Sequencing
For Northern blot analysis, purified RNA was electrophoresed on a 1% denaturing formaldehyde agarose gel, transferred to nitrocellulose, and hybridized as previously described.24 Analysis of the H19 level in RNA from rabbit tissues was carried out under lower stringency conditions. These Northern blots were hybridized in the presence of 40% formamide at 37°C and washed in 0.2x standard saline citrate at 37°C. DNA sequencing was performed by dideoxy chain termina-tion25 using the procedures recommended by the supplier. Analysis of DNA sequences and comparison with the GenBank data base were performed using software from IntelliGenetics, Inc., Mountain View, Calif.
Results

Identification of Two Developmentally Regulated Genes in Aortic SMCs and Comparison of Their Expression With Cytoskeletal Genes
Poly(A+) RNA from fetal (25-day gestation) rabbit aortic smooth muscle was isolated and used to prepare a cDNA library. To select fetal-specific genes, recombinant phage was plated at low density, the phage DNA was transferred to nitrocellulose, and duplicate lifts were screened with 32P-labeled cDNA from fetal rabbit aortic smooth muscle and from proliferating adult cultured SMCs. We used cDNA from proliferating adult cultured cells as the control probe to avoid obtaining cDNA clones associated with proliferation such as c-myc and c-fos. Twenty thousand recombinant plaques were screened initially, and from two rounds of screening, nine clones continued to preferentially hybridize to labeled cDNA from fetal aortic SMCs. Northern blot analysis revealed that only one cDNA clone, F-31, identified an RNA that was developmentally regulated in vascular smooth muscle ( Figure 1A , blot a). The 405-bp F-31 cDNA strongly hybridized to a 2.3-kb RNA in fetal aortic smooth muscle, and expression of this RNA was considerably decreased in newborn smooth muscle and was undetectable in 4-week-old adult aortic smooth muscle. IGF-I1 is believed to be a fetal growth factor and is present at high levels in many embryonic tissues.2 -29 We also examined whether this gene is developmentally regulated in vascular smooth muscle. The result, shown in Figure 1A , blot b, indicates that a 4.5-kb IGF-I1 mRNA was indeed expressed in fetal aortic smooth muscle but was not detected in adult smooth muscle. However, in contrast to F-31 expression, IGF-II was not detectable in newborn vascular smooth muscle.
Identification of Two Developmentally Regulated Genes in Aortic SMCs and Comparison of Their Expression With Cytoskeletal Genes
The mRNA levels of some of the major cytoskeletal and contractile proteins (actin, smooth muscle myosin heavy chain, and a-tropomyosin) were also examined in developing aortic smooth muscle ( Figure 1B ). The six members of the actin family are highly homologous and can be recognized by any one of the actin probes.311,31 Both /3-and y-actin species are encoded by mRNA of 2.1 kb; the a species are encoded by mRNA of 1.6 kb. Our /-actin eDNA probe detected both a 2.1-and a 1.6-kb RNA band, and the level of neither RNA was appreciably altered during development ( Figure 1B , blot a). By contrast, the smooth muscle myosin heavy chain eDNA identified a single 7.5-kb band that clearly increased with developmental maturation of the aorta In the second set of adult skin and lung RNA lanes in panel A, the Northern blot was exposed to film for 1 week instead of overnight at -70C.
Identification of Two Developmentally Regulated Genes in Aortic SMCs and Comparison of Their Expression With Cytoskeletal Genes
( Figure 1B , blot b). Densitometric analysis indicated a sixfold difference between fetal and adult smooth muscle. The a-tropomyosin eDNA specifically recognized two RNA species of 1.3 and 1.8 kb. Expression of the 1.8-kb RNA was unchanged, whereas expression of the 1.3-kb message was somewhat more prominent in adult smooth muscle.
F-31 Is Developmentally Regulated in Many Rabbit Tissues and Also in Cultured SMCs
We next examined whether the expression of F-31 was restricted to the aorta or whether it was expressed in other tissues. mRNA from a number of tissues was isolated and examined by Northern blot analysis. The results, shown in Figure 2A , indicate that fetal liver and heart tissue also contained high levels of F-31 RNA, but no F-31 was detected in the corresponding adult tissues. Results of the tissue distribution studies are summarized in Table 1 and indicate that most embryonic tissues contained high levels of F-31, whereas their adult counterpart contained low or undetectable levels of F-31. The highest level of F-31 was found in the placenta and in fetal muscle. Fetal esophagus, lung, liver, heart, and aorta also expressed relatively high levels of F-31, whereas fetal skin, kidney, and brain expressed less F-31. In the adult tissues, we could detect low levels of F-31 in the esophagus and lung, but all the other tissues contained either undetectable levels of F-31 or levels more than 20-fold lower than fetal aorta.
F-31 Is Developmentally Regulated in Many Rabbit Tissues and Also in Cultured SMCs
Interestingly, although the esophagus contained F-31 RNA, we were unable to detect any F-31 in the stomach and only extremely low levels in the small and large intestines (results not shown). There was not a simple correlation between relative levels of F-31 in fetal and adult tissues. For example, fetal liver, heart, and aorta expressed high levels of F-31, but we were unable to detect F-31 expression in the corresponding adult tissues. By contrast, fetal brain and skin contained very low levels of F-31, yet there were detectable levels in the adult. We next compared the expression of F-31 in aortic smooth muscle with cultured SMCs obtained from fetal, newborn, and adult aortas. The result, shown in Figure  2B , indicated that cultured fetal SMCs expressed a considerable amount of F-31, and densitometric analysis indicated that it was approximately threefold less than that expressed by fetal SMCs in vivo. Consistent with the in vivo data, newborn SMCs also expressed F-31, whereas adult SMCs did not ( Figures 1A, blot a, and 2B). We also examined the expression of IGF-II mRNA in cultured fetal, newborn, and adult SMCs. We found that IGF-II mRNA was detectable only in fetal cells ( Figure 5 and results not shown). This expression pattern was also consistent with that observed in vivo.
F-31 Is Developmentally Regulated in Many Rabbit Tissues and Also in Cultured SMCs
Complete Sequencing of 1.9-kb F-31 cDNA and Database Analysis Revealed That F-31 Is Homologous to the H19 Gene F-31 expression is developmentally regulated in a variety of tissues examined, suggesting that it may play an important role in a number of development and differentiation processes. Therefore, we further characterized F-31 by screening a rabbit placenta cDNA library for larger cDNA inserts. Using the initial 450 -bp cDNA insert isolated from the differential screening, 1.2-kb and 1.8-kb recombinant cDNA clones were isolated from a rabbit placenta random-primed cDNA library. Complete DNA sequence analysis and a subsequent search of the GENBANK data base revealed that F-31 is similar to a previously characterized, developmentally regulated gene called H19.20,32 Identity of rabbit F-31 with the human H19 gene was approximately 74% (Figure 3, left panel) . A similar degree of identity was observed with the mouse H19 gene (results not shown). Like H19, F-31 did not contain a large open reading frame (ORF). However, there were five ORFs that could potentially encode polypeptides of between 41 and 121 amino acids (Figure 3, right panel) . Since our cDNA did not contain the entire F-31 gene, we do not know the actual length of the first ORF (ORF A) or whether an appropriate initiating methionine is present. However, since this ORF is not conserved in the mouse or human H19 gene, its presence may not be relevant.32,33 The four AUG start sites (ORFs B-E) were found in the middle of the F-31 cDNA. Interestingly, these were the only four methionines in rabbit F-31, and corresponding translational start sites for all of them were also found in the human and mouse H19 sequences.32,33 The largest of these, ORF E, was from 1 base 1,159 to base 1,521 and could encode a 121-amino acid protein. The first eight amino acids in ORF E after the initiating methionine were identical between rabbit F-31 and the H19 gene (Figure 3, right panel) . However, the overall translated amino acid sequences of ORF E were poorly conserved between rabbit F-31, human H19, and mouse H19. Indeed, in both the human and the mouse H19 gene, this ORF extends only to 10 amino acids (Figure 3, right panel) . Interestingly, as shown in Figure 3 , right panel, the regions immediately adjacent to all four of the AUGs are well conserved between rabbit F-31 and the human and mouse H19 gene. However, similar to that seen for ORF E, the sequence identity was only maintained for a short stretch of amino acids. No sequence similarity was observed after about the first 10 amino acids (Figure 3 , and results not shown).
F-31 and IGF-II Expressions Are Regulated by the Growth State of Cultured Fetal SMCs
Since decreased expression of F-31 and IGF-I1 generally correlated with a decrease in SMC proliferation in vivo, it was of interest to examine whether the expression of these genes was also regulated by the growth state of cultured fetal SMCs. We first examined the morphology of these cells in culture and found that before reaching confluence, fetal cells were morphologically very similar to their adult counterparts (Figure 4 ). However, when fetal and adult SMCs were allowed to grow to a high cell density, a striking morphological difference was observed. Whereas adult cells became multilayered and acquired the "hill and valley" morphology typically associated with cultured SMCs, fetal cells remained monolayered and retained a spread and flat morphology (Figure 4 ). The finding that densityarrested fetal SMCs remained monolayered suggests that cell-cell contact may play an important role in the growth arrest of these cells.
F-31 and IGF-II Expressions Are Regulated by the Growth State of Cultured Fetal SMCs
We next examined the expression of F-31 and IGF-II as well as actin, smooth muscle myosin heavy chain, and a-tropomyosin in fetal SMCs cultured under three defined conditions. RNA was harvested from proliferating preconfluent cells, 7-day postconfluent cells, and 7-day postconfluent cells cultured for the last 3 days in medium containing 0.5% serum, 10`6 M insulin, and 5 ,ug/ml transferrin and analyzed by Northern blots. The results, shown in Figure 5 , indicated that levels of both the 2.1-and 1.6-kb actin mRNAs were unchanged but that there was a slight increase in smooth muscle myosin heavy chain level in postconfluent cells treated with 0.5% serum (observed in two of three experiments). We also examined a-tropomyosin expression and found that the 1.8-kb message was unchanged but that the 1.3-kb mRNA species increased slightly in postconfluent cells. We next examined the expression of F-31 and IGF-I1 and found that both were expressed in proliferating fetal cells but were low or undetectable in 7-day postconfluent quiescent fetal cells. However, in postconfluent cells treated with 0.5% serum, expression of F-31 but not IGF-II was again prominent ( Figure 5 ). Cultured adult cells under all three conditions do not express detectable levels of either F-31 or IGF-II ( Figure 2 and results not shown). These results indicate that in vitro expression of F-31 and IGF-II in fetal SMCs can be regulated by altered growth state and/or cell density.
Discussion
We have examined some of the molecular changes associated with the developmental maturation of blood vessels in late gestational and early neonatal life. By differential cloning, we have identified a gene (F-31) that was highly expressed in rabbit neonatal and newborn vascular smooth muscle but was undetectable in adult smooth muscle. In addition, we found that the growth factor gene IGF-II was also developmentally regulated in vascular smooth muscle. The mRNA levels of several genes that encode cytoskeletal and contractile proteins were also determined in neonatal, newborn, and adult SMCs. We found that total actin mRNA level and a-smooth muscle actin level were unchanged during development in the rabbit. This is in contrast to that previously reported for the rat, in which both an increase in a-smooth muscle actin mRNA level and a decrease in /3and y-actin mRNA levels resulted in a fourfold relative enhancement in a-actin mRNA level between 5-day-old neonate and 6-week-old rats.34 This discrepancy most likely reflects a species difference in actin mRNA regulation and is consistent with our previous in vitro studies indicating that altered expression of a-actin is not always a useful parameter to determine the proliferative state of SMCs.17 By contrast, the level of smooth muscle myosin heavy chain mRNA increased sharply in adult smooth muscle. This is consistent with recently published results and presumably reflects increased expression of the alternatively spliced SM2 form of this gene in aortic smooth muscle.9
Discussion
It is well established that IGF-II is expressed in a variety of fetal tissues and is especially prominent in cells of mesenchymal origin.28'29 That IGF-II is highly expressed early in development and has mitogenic activity in vitro has led to the suggestion that it has a role in fetal growth. 35 A direct demonstration that IGF-II may play an important role in fetal growth was recently provided by transgenic studies in which mice carrying only one allele of the functional wild type IGF-II exhibited a 10-fold decrease in IGF-II mRNA expression and a 40% decrease in animal size. 36 The developmental regulation of IGF-II expression in vascular SMCs has not been examined previously. We have shown that fetal rabbit vascular SMCs expressed a major IGF-II RNA transcript of 4.5 kb that was bennettsteve@example.net. Furthermore, cultured fetal SMCs continued to express IGF-II, but its expression was downregulated by high cell density-associated growth arrest. Our results are consistent with the possibility that IGF-II may have some autocrine role in the proliferation of fetal vascular SMCs. However, we were unable to detect IGF-Il expression in neonatal SMCs, and since these cells continue to divide in vivo, although at a slower rate, other growth factors must also be involved in the continued proliferation of these cells.
Discussion
We have used differential screening to identify a second gene (F-31) that is expressed in fetal SMCs but not in adult cells. Unlike IGF-II, the expression of F-31 was reduced but clearly present in newborn SMCs. This expression pattern more closely parallels that of the proliferative pattern of SMCs.5-7 We also found that F-31 was expressed in proliferating fetal SMCs in culture but that its expression was turned off in postconfluent cells. However, when the postconfluent cells were cultured in the presence of low serum media, there was reexpression of F-31. One explanation for these findings is that F-31 may have more than one role in SMC growth and maturation. F-31 is most likely the rabbit homologue of H19.2t) 32 This is based on a nucleotide sequence identity of approximately 70%, a very similar developmental expression pattern, and a curious lack of a major translational ORF. The H19 gene encodes an RNA polymerase IL product that is expressed at high levels in most mouse embryonic tissue of endodermal and mesodermal origin and is dramatically bennettsteve@example.net That H19 has a crucial role during development has been demonstrated by the finding that the introduction of the H19 gene in mice caused prenatal lethality in late gestation.37 At present, the function of H19 is unknown, although there is good evidence to suggest that its function may not be associated with translation to a polypeptide product.32 H19 has also been independently isolated by investigators interested in genes activated during myoblast differentiation (MyoH) and during embryonal carcinoma cell differentiation and presumably has a role in the differentiation of these cellS 33, 38, 39 Our findings are consistent with the notion that F-31 has an important role in vascular smooth muscle development. We cannot absolutely rule out the possibility that we have isolated a separate gene closely related to the H19 gene. There does appear to be some discrepancy between the adult tissue expression pattern we have observed and that reported for H119 in the mouse.33,41) In the adult mouse, skeletal muscle contains the most abundant amount of H19, with lower levels observed in the heart, lung, thymus, and liver and none detected in the brain. By contrast, we found that the level of F-31 was approximately four times higher in the adult lung than in skeletal muscle. Furthermore, we were able to detect F-31 in the adult brain but not in the heart or the liver. Whether these differences reflect expression differences between two closely related genes or are merely due to the species examined is presently unclear, since we were unable to directly measure H19 expression in the adult tissues because of poor cross hybridization of the mouse H19 gene with rabbit.
Discussion
Finally, it should be emphasized that we have identified two developmentally associated in vivo changes in vascular smooth muscle that are maintained in vitro between fetal and adult SMCs. This result and the high density-dependent distinct morphology observed between cultured fetal and adult SMCs indicate that these cells are able to maintain their separate phenotypes in vitro. A better understanding of such differences between cultured fetal and adult cells may provide additional insights into the developmental maturation of SMCs. In conclusion, we have identified F-31/H19 and IGF-IL as two genes that are developmentally regulated in vascular SMCs. These genes provide two parameters to examine the hypothesis that SMCs induced to proliferate under pathological conditions may recapitulate earlier developmental events.

{'paper_id': '24302008', 'title': 'Identification and characterization of developmentally regulated genes in vascular smooth muscle cells.', 's2_url': 'https://api.semanticscholar.org/CorpusID:24302008', 'source': 'S2ORC'}