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Physiol. Genomics 25: 250-262, 2006. First published January 17, 2006; doi:10.1152/physiolgenomics.00231.2005 Free Article
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Received 13 September 2005; accepted in final form 13 January 2006.
Physiological Genomics 25:250-262 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Gene expression in ductus arteriosus and aorta: comparison of birth and oxygen effects

Mario Costa 1,*, Silvia Barogi 2,3,*, Nicholas D. Socci 4, Debora Angeloni 2,3, Margherita Maffei 5, Barbara Baragatti 2,3, Chiara Chiellini 5, Emanuela Grasso 2 and Flavio Coceani 2,3

1 Institute of Neuroscience, Consiglio Nazionale delle Ricerche (CNR), Pisa, Italy
2 Scuola Superiore S. Anna, CNR, Pisa, Italy
3 Institute of Clinical Physiology, CNR, Pisa, Italy
4 Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, New York
5 Dulbecco Telethon Institute and Department of Endocrinology, University of Pisa, Pisa, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ductus arteriosus (DA) closure is initiated by oxygen rise postnatally and progresses in two, functional-to-permanent, stages. Here, using GeneChip Arrays in rats (normoxic and hyperoxic fetus, normoxic newborn), we examined whether oxygen alone duplicates the birth process in affecting DA genes. In addition, by comparing DA with aorta (Ao), we identified features in postnatal gene profile marking transitional adjustments in a closing (DA) vs. a persistent (Ao) vessel. We found changes in neonatal DA denoting enhanced formation and action of the constrictor endothelin-1 (ET-1). Likewise, ANG II type 1 receptor was upregulated, and the compound was a constrictor. Conversely, relaxant PGE2 became less effective. Among agents for functional closure, only ET-1 was affected similarly by oxygen and birth. Coincidentally, neonatal DA showed enhanced contractile drive with upregulation of Rho-Rho kinase and calcium signaling along with downregulation of contractile proteins. The latter effect was shared by oxygen. Changes denoting active remodeling were also seen in neonatal but not hyperoxic fetal DA. Ao, unlike DA, exhibited postnatal variations in noradrenergic, purinergic, and PGI2 systems with opposing effects on vasomotion. Contraction and remodeling processes were also less affected by birth, whereas lipid and glucose metabolism were upregulated. We conclude that several agents, including ANG II as novel effector, promote functional closure of DA, but only ET-1 is causally coupled with oxygen. Oxygen has no role in processes for permanent closure. Functional closure is associated with downregulation of contractile apparatus, and this may render neonatal DA less amenable to tone manipulation. Conceivably, activation of metabolism in neonatal Ao is a distinguishing feature for transitional adaptations in the permanent vasculature.

ductus closure; postnatal programming; fetal and neonatal physiology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE CARDIOVASCULAR SYSTEM undergoes major changes during the transition from intra- to extrauterine life, and among these closure of a shunt, the ductus arteriosus (DA) connecting the pulmonary artery with the aorta (Ao), has received much attention for its immediacy, impact on hemodynamics, and feasibility of therapeutic manipulation (30). Preparation for this event begins early in gestation with development of a prominent muscle layer within the vessel wall, progresses with formation of intimal cushions in species whose duct exceeds a critical size (e.g., humans but not rodents), and culminates at birth with an abrupt contraction and, ultimately, with structural changes leading to obliteration (3). Several factors have been implicated in this sequence, with the action of vasoactive agents for the initial functional closure being intertwined with the remodeling process for permanent closure. Specifically, at birth there is synergism between activation of a contractile mechanism, conceivably involving endothelin-1 (ET-1) (11, 19, 33) and a voltage-gated K+ (Kv) channel (19, 24) as effectors, and removal of a tonic relaxation from blood-borne PGE2 (11, 13). Critical for the onset of this constriction is the postnatal rise in blood oxygen tension. Coincidentally, as the vessel narrows, a series of structural alterations begins in which several factors (adhesion factors, vascular endothelial cell growth factor, fibronectin, laminin, cytokines, certain apoptosis-linked molecules) (7–9, 16, 20, 22, 29, 35) have already been implicated. Changes comprise a progressive intimal thickening secondary to endothelium ingrowth and muscle cell migration, media remodeling through a combination of muscle proliferation/dedifferentiation and apoptosis, and, finally, apposition of endothelial lining with the ensuing obliteration of the vessel lumen. Although the essential features of this sequence are known, no information is available on the actual role being played by oxygen in its individual steps. In particular, from the available data it is not clear whether oxygen simply serves as trigger for functional DA closure or whether certain aspects of the subsequent vessel remodeling are also directly connected to the action of this agent. An additional, broader question is how transitional adjustments at birth condition the gene expression profile in DA vis-à-vis a vessel, such as Ao, that remains patent. Both issues were addressed in our investigation. Elucidation of these points is important not only to better characterize oxygen-linked events but also to fully define vascular mechanisms being affected by the birth process. In addition, knowledge of the gene profile in the perinatal vasculature may serve as a reference for any investigation of a developmental link in cardiovascular diseases of the adult.

Using GeneChip arrays, we confirmed the importance of changes in the ET-1 and PGE2 systems for functional DA closure but, at the same time, identified in ANG II a potential additional effector. Only ET-1 function, however, was linked with oxygen. As anticipated, DA exhibited a cohort of gene changes denoting active remodeling. Among these, an unexpected feature was the downregulation of contractile proteins appearing as a sign of oxygen action. The remainder of the remodeling process could instead be connected causally with signals deriving from shear stress on the luminal surface of the narrowing vessel and the subsequent intramural hypoxia. Contrary to DA but consistent with tone regulation, Ao showed postnatal activation of vasomotor mechanisms with opposing effects. Coincidentally, there was upregulation of genes for energy metabolism, manifesting collectively the demands of a rapidly growing and differentiating organ.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of rat DA and Ao.
Long-Evans rats were mated for 24 h, and the end of this period was marked as day 0 of pregnancy. Near-term fetuses (gestational age 19 days) were delivered by cesarean section under chloral hydrate anesthesia (35–70 mg/100 g body wt ip) from a normoxic or hyperoxic dam, whereas newborns were used 3 h after vaginal delivery. Pregnant rats were made hyperoxic to reproduce in utero the neonatal condition with the attendant DA constriction (11). They breathed 100% oxygen inside a box for 3 h, and throughout this procedure they were anesthetized with chloral hydrate and kept warm with water-filled bags at 39°C. Control experiments confirmed that blood PO2 values for the fetuses from hyperoxic dams were within the range of those for newborns (see Supplemental Table 4, available at the Physiological Genomics web site).1 All animals were killed by cervical dislocation and, to isolate the DA and Ao (arch and thoracic sections), were secured inside a chamber filled with ice-cold Krebs medium through which was bubbled a gas mixture with 2.5% (fetus) or 12.5% (newborn) oxygen (1). Special care was taken to obtain blood vessels free of any adhering tissue.

Total RNA preparation.
Specimens from normoxic (DA, Ao) and hyperoxic (DA) fetuses and newborns (DA, Ao) were pooled to obtain five distinct groups (n = 52–142/group and 20–25/group, respectively, for DA and Ao) and were homogenized in TriPure isolation reagent (Roche, Indianapolis, IN). The procedure followed a published protocol (1), and RNA yield was measured spectrophotometrically. Quality of the material was assessed with ethidium bromide staining on formaldehyde gel.

DNA microarray.
Analysis of mRNA expression was performed with an Affymetrix GeneChip Array (rat genome U34) comprising three distinct gene chips for a total of ~24,000 entries. Biotin-labeled cRNA was prepared from 10 µg of total RNA, as recommended by the manufacturer. Hybridization, washing, and staining of the gene chips were carried out in an Affymetrix oven and fluidics station. They were then scanned with a Hewlett-Packard confocal laser scanner, and images were analyzed with Affymetrix Genechip 3.3 software. This software gave intensity data normalized against the mean fluorescence intensity of each array. Initial analysis and pair comparisons were made with a twofold cutoff, a change P value of <0.0025, and a detection P value of <0.05. After log transformation, a baseline value of 1 was used as a reference in determining for each gene the ratio in level of expression between newborn and fetus (1 signal log ratio = 2-fold change). Subsequent clustering of genes was performed in two ways. In the first method (algorithm I) (37), any native enrichment, possibly indicative of a specific function, was ascertained in each of the four vessel-age combinations (i.e., DA/Ao vis-à-vis fetus/newborn) relative to the others. This was done at increasing fold-change cutoffs (1.3, 1.6, 2, and 2.3 log ratio values) to vary the stringency of the test. Every condition was compared with the other three, using the standard fold-change method, and only genes that had passed the fold-change and P-value cutoffs (see above) in all comparisons were added to the list. In the second mode, limited to DA (algorithm II), overlapping changes were evidenced in the comparisons of the hyperoxic fetus with the normoxic newborn, using the normoxic fetus as a reference for both. Genes were clustered into four groups based on the sign of the two comparisons, and the attendant coincidence, or lack of coincidence, of changes in the hyperoxic fetus vs. the newborn (up/up, down/down, down/up, up/down) provided a possible measure of the specific contribution of oxygen to the transitional adjustments at birth. The cutoff level was, in this case, lowered from 2 to 1.5. and the baseline value became accordingly 0.6 (0.6 signal log ratio = 1.5-fold change).

The complete set of data is available in the GEO database (accession no. GSE3290).

Quantitative RT-PCR.
Genes selected for this analysis (Myl2, Agtr1a, Agtr2, Igf1, Pf4, Pparg) exemplify functional categories relevant to our findings (see RESULTS). In addition, some of them (Pparg, Igf1) are noteworthy for their coordinating function. DNAse I (Roche)-treated total RNA (2 µg) was reverse-transcribed with 1 U of ThermoScript RT (Invitrogen, Carlsbad, CA) in the presence of random hexanucleotide primers according to the manufacturer’s instructions. Quantitative RT-PCR (QRT-PCR) reactions (40 cycles) were performed on an ABI Prism 7700 instrument (Applied Biosystems, Foster City, CA), using TaqMan Universal PCR Master Mix (Applied Biosystems). Primer sequences for Pparg, Igf1, Agtr1a, Agtr2, and cyclophilin B (internal standard) were obtained from an online library (Applied Biosystems). The remaining sequences were designed with File-Builder software (Applied Biosystems) and were as follows: Myl2 (exon 4): forward primer 5'-ACT GTG TTC CTC ACC ATG TTT GG, reverse primer 5'-CCT TGA AGG CGT TGA GAA TGG, probe (Fam) 5'-CCG GGT CAG CTC CTT TA; Pf4 (exon 3): forward primer 5'-GCC GGT CCA GGC AAA TTT TG, reverse primer 5'-CAA AGC AGG ACC CCA CTG T, probe (Fam) 5'-CCC CAG CTC ATA GCC AC. Gene expression was quantified with the comparative cycle threshold method using cyclophilin for normalization, and results were obtained in triplicate.

Mouse DA response to ANG II.
The mouse, sharing with the rat up to 94% of the genome and the timing of DA closure (29), was used because of the availability of reference data on vasoactive agents (1). Near-term fetal C57BL/6 animals were delivered by cesarean section under halothane anesthesia and were killed by cervical dislocation. The DA was mounted in an organ bath, as previously described (1), and mechanical tension was recorded isometrically at 2.5% or 12.5% oxygen to mimic, respectively, the fetal or the neonatal condition. ANG II was tested in sequential doses (0.01 nM-10 µM, final in bath), using 10-fold increments, and effects were measured by the fractional change from baseline. Data are expressed as means ± SE.

Surgical procedures and experimental protocols were approved by the Animal Care Committee of the Ministry of Health.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of vessel- and age-linked markers.
Using algorithm I (see METHODS), we found distinctive sets of genes depending on vessel and age of the animal (DA, fetus: 4; DA, newborn: 10; Ao, fetus: 14; Ao, newborn: 20) (Table 1). DA showed a preferential expression of genes for contractile proteins in the fetus, whereas genes for vasomotor control (Rhob, Pde4b) and tissue remodeling (cytoskeleton/matrix components, elements of the immune complex) became predominant in the newborn. Quite novel among postnatal changes in DA was the appearance of galanin (Gal), which may contribute, in a hitherto undefined manner, to the process of closure. The fetal Ao, on the other hand, was characterized by a diversified transcriptional profile comprising genes for blood-borne tissue constituents, growth (Gas2), and neurotransmission (Chgb). No such diversification was noted in the neonatal Ao, where, in line with the requirements of a growing organ, most marker genes related to lipid and glucose metabolism and energy expenditure control. Transcripts for tissue modeling and redox state regulation made up the remainder of this pool.


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  		Table 1. Hierarchical gene clustering in DA vs. Ao before and after birth

 
Effect of oxygen vs. birth on DA gene expression.
Considering that oxygen is the prime trigger for DA closure, we investigated next whether its action on gene expression mimics that being exerted naturally by birth. A total of 180 entries (Supplemental Table 1) satisfied the criteria of algorithm II (see METHODS), and among these 110 showed similar changes, either upward (33 entries) or downward (77 entries), under the two conditions. In those instances in which this coincidence was missing, we found in the main downregulation by oxygen and upregulation by birth (64 entries). The opposite relationship was also seen, but it was limited to six entries.

Table 2 lists genes from this comparison identifiable with certain functional categories. Within the structure group (group 1), genes for cell architecture, the contractile apparatus, and blood-borne tissue constituents were, in general, similarly affected by the two conditions. Peculiar in these events is the reduced expression of contractile proteins in response to oxygen alone, considering the role assigned to this agent in the functional closure of the vessel. Conversely, several genes for tissue remodeling (Tgm2, Cast), cell growth/motility (fn-1), and cell reactivity (Cav, Thbd, Pecam1, Itga8, Emp1) were downregulated by oxygen and upregulated by birth. The same divergence was found in a host of genes for signaling factors (group 2) pertaining to the viability of cells (Rgs5, Mig-6, luc7l2), some special functions such as growth and the response to stress (Cic, Anxa3, Mapkapk 2, Dusp 1, ankyrin repeat and SOCS box-containing protein 2), and muscle contraction (Ppp1r12a, Calm1). Equally divergent were responses of the gene for cAMP phosphodiesterase (Pde4b). Considering the importance of Pde4b in the termination of PGE2 effects, this finding implies that only postnatally suitable conditions take place for curtailment of DA relaxation by PGE2 (4). Indeed, Pde4b is a selective marker for neonatal DA (see above). A better coincidence was noted with genes for transcription factors and receptors/carriers. Specifically, Gata2, with its potential positive control on transcription of the ET-1 gene, was upregulated in the hyperoxic fetus as in the newborn. Similarly consistent, albeit in the opposite direction, was the response of the ETB receptor subtype of ET-1 (Ednrb) mediating vasodilatation (10). Collectively, these findings indicate that oxygen alone may promote only one of the postulated mechanisms (i.e., the ET-1 system) for functional closure of DA, whereas it is unable to exert a positive effect on the host of factors for permanent closure. Particularly significant in this context is the downregulation of genes for vascular mechanotransduction (Cav, Pecam1, Itga8), implying that shear stress from oxygen constriction does not initiate any remodeling cascade.


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  		Table 2. Gene expression changes in DA from hyperoxic fetus vs. newborn

 
Genes for growth factors (group 3) were downregulated by oxygen (Ghrh, Bmpr1a, Smoc1, Igf1 and Igf2, Fgf18) and, with some exceptions (Ghrh, Bmpr1a), also by birth. Downregulation of the neuropilin gene (Nrp1) under either condition (see group 2) is consistent with this finding. Two of three genes for angiogenetic factors (Cyr61, Nogo, but not Crim1) behaved in the same way, whereas the single antiangiogenesis gene (Pf4) was consistently upregulated. On the other hand, regardless of their sign of action, genes for cell proliferation and apoptosis were downregulated in the oxygen-exposed fetus and upregulated in the newborn. The latter finding reaffirms the concept, emerging here, that oxygen alone cannot promote the combination of opposing, growth vs. apoptosis, processes necessary for DA remodeling and, ultimately, for permanent closure.

The metabolic system (group 4) comprised assorted genes for cell function showing upregulation under both conditions or downregulation with one (oxygen) and upregulation with the other (birth). Noteworthy in the former category was the cytochrome b5 transcript (Cyb5) for its potential facilitation of cytochrome P-450 (CYP450)-based monooxygenase reactions. Otherwise, a pool of genes connected with lipid and glucose metabolism, including the transcript for a fatty acid synthase, were uniformly downregulated. DA, in the latter respect, differs sharply from Ao.

Only few genes belonged to the immune system (group 5) and, regardless of their action, were affected similarly by oxygen and birth. Conversely, the MICAL 2 gene, encoding a protein with a monooxygenase domain and possible redox-sensitive function (34) (group 6), responded unevenly to the two conditions.

Gene profile in DA and Ao: postnatal vs. prenatal.
The impact of birth greatly exceeds that emerging from a comparison with the oxygen effect. We found a large cohort of genes to be affected, and, despite differences between DA and Ao, most changes could be arranged into broad categories (Supplemental Tables 2 and 3). In the case of the structural component (Supplemental Tables 2 and 3, group 1), both DA and Ao showed a combination of upregulatory and downregulatory variations in the expression of genes encoding membrane proteins, protein processing systems, and a vast assortment of cytoskeleton/matrix constituents. Upregulation encompassed cytokine-induced proteins especially in Ao, distinct structural moieties (Des, Kb4, cell surface glycoproteins, Olfml25), and factors regulating cell growth/motility or the ability of cells to interact with their immediate environment. Conversely, certain genes (Tubg, Fbn2, Marcks, Cdh1, Krt1–19, ribosomal subunits particularly in DA) were downregulated. Collectively, these findings point to a structural rearrangement taking place in both vessels. Indeed, in accordance with this process was a combination of upward and downward changes in genes regulating protein processing intracellularly (e.g., ubiquitin complex) or matrix turnover extracellularly (e.g., metalloproteinase). Some responses, however, were specific to either vessel. Of note in DA was the upregulation of the P-selectin gene (Selp) along with genes for cell growth and migration (Tnn, mena protein, fibulin-1, osteopontin). Potentially important for the timing of DA closure was also the upregulation of a carboxypeptidase (Cpa3), i.e., an enzyme for breakdown of the constrictor ET-1. Conversely, some structure-related genes were downregulated postnatally (actinin, Ugcgl1, STAB1, Phactr2). Modifications specific to Ao had also opposing sign and comprised upregulation of structure-linked genes (Jph2, Mtap6, Grifin) with genes for cell growth (Ler3)/differentiation (Popdc2) and downregulation of one of the allied genes (similar to CG3284-PA).

No such diversity was noted with genes for the contractile apparatus because their expression varied uniformly, either downward (DA) or upward (Ao), in the immediate neonatal period.

Few changes in both vessels concerned blood-derived factors. After birth, hemoglobin had the expected shift from fetal to adult variant, {alpha}-fetoprotein (Afp) was downregulated, and a mixed group of transcripts for carriers, complement-related molecules, and immunoglobulins was unevenly affected. Quite striking, however, was the upregulation in Ao of genes for the hormone adiponectin (Acdc), promoting fatty acid and glucose metabolism, and the protease adipsin (Adn). Both these factors are secreted by adipocytes; however, Acdc may also be formed elsewhere (28).

Transcription factors showed a complex pattern of transitional variations, with some distinctive features (Supplemental Tables 2 and 3, group 2). Significant was the upregulation in DA of Gata2 favoring ET-1 function. However, another member of the same family, Gata4, linked to cardiovascular development and differentiation, presented an opposite change. Characteristic of Ao was the upregulation of peroxisome proliferator-activated receptor-{gamma} (Pparg) with its expected positive control on Acdc and a cohort of genes for lipid and glucose metabolism (see below). Both vessels, on the other hand, shared the upregulation of BTE binding protein (Bteb1), i.e., a factor interfering with the expression of a CYP450 gene (CYP1A1) of potential importance for vasoregulation. CYP1A1 may, in fact, contribute to arachidonic acid (AA) {omega}-hydroxylation (6). Several genes for the Krüppel-like family (Klf) of transcription factors were also found, including the endothelium-based Klf4 (upregulated) and the muscle-based Klf5 (downregulated), respectively, in DA and Ao. Klf4 is activated by shear stress (14) and, together with allied genes being similarly upregulated (Cav, VE-cadherin, Itga, Pecam1; see above), may initiate the expected remodeling in the neonatal DA. Downregulation of aortic Klf5, on the other hand, accords with the regression of this particular gene with cell maturation (14).

A mixed group of signaling factors was also affected by birth. Noteworthy was a set of coordinated changes in transcripts for the eicosanoid system and calcium regulation. In DA, there was upregulation of phospholipase A2 (Pla2g2a), with its inhibitor lipocortin (Anxa1), and the AA-linked 12S-lipoxygenase (Alox15). Among the calcium-linked events was the upregulation in DA of a cluster of genes including the Rho-Rho kinase signaling system for contraction (Rho B/ROK-{alpha} complex), Calm1 with an associated modulator (PEP-19), and the membrane transducing factor copine. Concomitantly, the phospholamban gene (Pln) was downregulated. Furthermore, genes for calcium-binding proteins were either upregulated (calgranulin A, MRP14) or downregulated (Vsnl1). The same dual response was seen with members of the synaptotagmin (Syt) family of calcium-sensing proteins (upregulation: Syt4; downregulation: Syt1). Collectively, findings in the neonatal DA point to a modified calcium homeostasis together with an enhanced drive toward contraction. Significantly, the Rho-Rho kinase system may also promote transformation of the muscle cell phenotype from synthetic to contractile (36). A comparable, albeit more limited, set of changes occurred in Ao, where, in particular, some of the genes for calcium signaling were undetectable. However, Ao, unlike DA, presented an upregulated PGI2 synthase gene (Ptgis).

Both DA and, to a larger degree, Ao were endowed with genes encoding carrier molecules and assorted receptors for vasoactive agents, cytokines, and growth factors. Particularly significant and novel was the differential effect of birth on genes for vasomotor mechanisms. DA showed upregulation of the ANG II type 1 receptor gene (Agtr1a) together with downregulation of Ednrb, and the two changes combined conceivably have a bearing on functional closure. Ao was, in contrast, characterized by the lesser expression of a Agtr1a-related receptor (Agtrl1) and by the upregulation of purinergic (P2rx1, P2ry2) and {alpha}1-adrenergic (Adra1d) receptors. The gene profile for receptors to chemokines/cytokines and growth factors combined upregulatory and downregulatory variations. In addition, peculiar to Ao was the upregulation of a transcript for the adipocyte water channel (Aqp7).

DA and Ao shared many genes for growth and angiogenesis undergoing transitional changes (Supplemental Tables 2 and 3, group 3). However, whereas growth-related genes showed a combination of upregulation and downregulation in both vessels, angiogenesis-related genes presented this dual pattern only in Ao. Specifically, we found a cohort of gene transcripts in both vessels favoring growth through upregulation or, conversely, interfering with it through a set of reciprocal changes. In addition, there were specific genes in each vessel exerting a positive (DA: EGF-like growth factor; Ao: Lpin1, Bmp6, Akt1, Ctgf) or a negative (DA: Itr; Ao: Nog, VEGD) influence on growth. A similar combination of proangiogenetic (upregulation: Cyr61) and antiangiogenetic (upregulation: Pf4, thrombospondin; downregulation: Bai3, Angpt2) responses was seen in the neonatal Ao. Conversely, in the DA the gene transcript for the proangiogenetic Cyr61 was downregulated, and genes being upregulated had an antiangiogenetic action (Pf4, thrombospondin). Indeed, in DA angiogenesis might be favored only by downregulation of the antiangiogenetic gene chondromodulin-1 (Chm-1). This complex pattern, conceivably reflecting events in diverse cell populations in both vessels, attests to an active structural rearrangement that in the case of DA underlies the evolution/involution sequence for permanent closure but in the case of Ao ensures the acquisition of the adult asset. A similar reasoning applies to the host of gene changes relating to proliferation and demise of cells (see below).

We also found in the neonatal DA and Ao alterations in genes concerning specifically the regulation of the cell cycle and apoptosis. Many of these were vessel specific. In the case of DA, birth elicited opposing influences on cell proliferation, with either action resulting from reciprocal modifications in positively and negatively acting genes. Accordingly, the hyperplastic drive derived from upregulation and downregulation of proliferative (Cdc25B) and antiproliferative (p57Kip2, Mll5, Tmpo, Phb, similar to SKB1 homolog) genes, respectively. This was countered by an equivalent combination of upward (antiproliferative: Pmf31, Myd116, Btg2, Txnip, Tieg, Enc1) and downward (proliferative: Ccna, Cdc20, Ccnd) changes. No such arrangement was seen with apoptosis, and changes in the neonatal DA denoted the greater impact of proapoptotic (upregulation: Nfkbia, TDAG51, Stk17b, Tieg) over antiapoptotic (upregulation: Bnip3; downregulation: DIO1) influences. The pattern was different in Ao, with the single proliferative gene (similar to cell cycle protein p55CDC) being downregulated and the larger pool of antiproliferative genes showing a combination of upregulatory (Cdkn1a, Hrasls3, Ccnc, Btg2, Tob1, Gadd45, Txnip) and downregulatory (Gas2, p57Kip2, Tmpo) changes. The apoptosis system, on the other hand, presented a host of opposite changes involving both positively acting (upregulation: Cidea, NDG2, Nfkbia; downregulation: Cideb) and negatively acting (upregulation: rIAP1; downregulation: similar to HSCO protein) genes. On balance, however, the antiapoptotic influence conceivably prevailed, in view of the singularly striking upregulation of the rIAP1 transcript.

A separate pool of genes, particularly prominent in Ao, related to metabolism (Supplemental Tables 2 and, group 4). Some of these, being upregulated in either (DA: Sat) or both (glnA) vessels, are concerned with tissue growth and remodeling. Other genes, occurring specifically in Ao, subserve diverse aspects of cell metabolism, and among these the upregulation of an enzyme (Gdp1) for oxidative metabolism was particularly striking. Also noteworthy in Ao was the upregulation of cytochrome b5 (Cyb5), possibly facilitating CYP450-based monooxygenase reactions. The latter finding, together with the upregulation in the same vessel of a cytosolic epoxide hydrolase (Ephx2) and the possible modulation of {omega}-hydroxylation reactions by Bteb1 (see above), points to a concerted rearrangement of fatty acid metabolism and its active products. However, distinctive for Ao was the upregulation of a cohort of genes controlling diverse steps in lipid metabolism. Collectively, these changes ensure availability of free fatty acids and their utilization by cells in an organ that is growing and is bound to remain viable. Congruent with this arrangement was the selective upregulation of Pparg and Acdc (see above). Equally specific to Ao was the upregulation of the uncoupling protein Ucp1 with its control on energy expenditure. In fact, energy homeostasis appeared to be finely regulated in Ao, because there was, concomitantly, downregulation of a second uncoupling protein (Ucp2) and upregulation of Cidea (see above), a proapoptotic factor inhibiting Ucp1 (21). Metabolic steps for glucose were also activated in Ao, along with fatty acid turnover and utilization. In contrast, genes for cholesterol biosynthesis were downregulated in both vessels.

Genes for immune receptors and related factors were also evident in DA and, to a lesser degree, in Ao (Supplemental Tables 2 and 3, group 5). They were unevenly affected by birth, but, on balance, upregulation prevailed. Equally enhanced were components of the complement complex (see above), whereas certain chemotactic agents exhibited divergent changes in DA. Conceivably, these postnatal events reflect, at least in part, a remodeling process.

Characteristic of the two vessels were genes identifiable with neural tissue (Supplemental Tables 2 and 3, group 6). They belonged, in the main, to structural proteins and development-linked factors and were variably affected by birth. Most significant in DA was the upregulation of Gal, presenting itself as a distinct marker (see above), whereas Ao showed downregulation of genes for norepinephrine synthesis and release (Ddc, Dbh, Chgb, STX1B). The latter finding, with the concomitant upregulation of the {alpha}1-adrenergic receptor (see above), implies a rearrangement of noradrenergic innervation through the transitional period at birth.

Uniformly upregulated were genes belonging to the redox/oxygen adaptation system (Supplemental Tables 2 and 3, group 7). They formed in both vessels a diversified group comprising on one hand defense or adaptive factors for the abrupt increase in tissue oxygenation at birth (Ca3, Sod2 and 3, Hdh, Mgst1) and on the other factors (Hspb1, Hspb1a) denoting a situation of stress. Apparently incongruent was the upregulation in DA of a Hif-related gene (Rtp801), particularly vis-à-vis Hif1a downregulation in Ao. However, this paradoxical response of DA likely results from tissue hypoxia consequent to forceful constriction.

QRT-PCR validation.
To validate findings with the GeneChip Array analysis, we performed QRT-PCR on certain genes exemplifying postnatal adjustments of fetal DA and Ao as well as responses of the fetal DA to oxygen. Two of these genes were also distinctly expressed in either DA (Myl2) or Ao (Pparg) (see Table 1). Results coincided with the microarray data (Fig. 1). The ANG II type 1 receptor increased its mRNA expression in DA through transition from the fetus to the newborn (1.7-fold), and this effect was not mimicked by oxygen, which tended to produce a change in the opposite direction (1.4-fold decrease) (Fig. 1A). A falling trend was also seen in Ao when comparing the prenatal with the postnatal condition (1.6-fold decrease) (Fig. 1A). On the other hand, the ANG II type 2 receptor, which could not be detected by microarray analysis, was not affected by birth in DA and decreased slightly in Ao (1.4-fold) (Fig. 1B). Myl2 mRNA expression abated in DA from the fetus to the newborn (8-fold) or on exposure to oxygen (2.4-fold), whereas the reverse occurred in Ao (4.1-fold increase) (Fig. 1C). Levels of Igf1 mRNA in the fetal DA decreased on exposure to oxygen (1.7-fold) and, to a larger degree (3.5-fold), through birth. An even greater fall (11-fold) from the fetal value occurred in the neonatal Ao (Fig. 1D). In contrast, Pf4 mRNA increased at birth 3.2- and 5-fold, respectively, in DA and Ao, and a similar trend (1.7-fold increase) was noted in DA in response to oxygen (Fig. 1E). Likewise, Pparg mRNA rose markedly (21-fold) from the fetal to the neonatal Ao, whereas under the same conditions the DA signal, which had been below detection with microarray, showed only a modest increase (2-fold) (Fig. 1F).


Figure 1
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  		Fig. 1. Quantitative RT-PCR (QRT-PCR) on selected genes from rat ductus arteriosus (DA) and aorta (Ao). Specimens were obtained from the fetus at 19 days of gestation (F), from the newborn at 3 h of age (N), and, in the case of DA, also from the fetus of dams made hyperoxic for 3 h (F/O). Levels of transcripts are expressed in arbitrary units after normalization by the cyclophilin control.

 
DA response to ANG II.
ANG II contracted the mouse DA in a concentration-dependent fashion, starting from a threshold of ~1 nM (Fig. 2A). The contraction developed quickly and, after reaching a maximum, equally rapidly abated to the original baseline, despite the continued presence of the substance in the bath (Fig. 2B, top). Occasionally, the reversal was not complete and a secondary, smaller contraction ensued (Fig. 2B, middle). The magnitude of responses was the same at 2.5% and 12.5% oxygen (Fig. 2A). However, at the higher oxygen concentration, the contraction was followed by a relaxation (Fig. 2B, bottom). This relaxation, unlike the contraction, did not develop dose-dependently (Fig. 2A) and could reflect an activation of the complex ANG II type 2 receptor/nitric oxide effector (17). The alternative possibility of a PGI2 effector function is unlikely, because the compound is not very active on DA (30).


Figure 2
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  		Fig. 2. Effect of ANG II on the DA from fetal mouse. A: concentration ([ANG II]) -response curve at 2.5% vs. 12.5% oxygen (n = 4 and 5). Values are means ± SE (error bars not visible are within the size of symbol). Note that a biphasic response, with a contraction (closed symbols) being followed by a relaxation (open symbols), was seen only at the higher oxygen concentration. B: representative responses to 1 µM ANG II at 2.5% (top and middle) and 12.5% (bottom) oxygen. Application took place between arrows. Scale bars, 50 mg for 5 min (100 mg wt = 0.98 mN)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
DA and Ao showed several differences in their postnatal vs. prenatal gene profile. Transcripts pertaining to vasoregulatory mechanisms presented a distinct pattern, with DA expressing an enhanced contractile drive through appropriate modifications in the ET-1- and PGE2-linked processes and Ao exhibiting instead a combination of opposing influences over tone through the involvement of noradrenergic, purinergic, and PGI2 systems. Accordingly, a cluster of genes for contraction, comprising the Rho-Rho kinase system and allied calcium signaling elements, was prominently upregulated in DA. A novel feature was the selective, birth-related upregulation of the ANG II type 1 receptor in DA along with the demonstration of the constrictor effect of the agent. This finding points to a contribution of ANG II to DA closure, a possibility considered in the past (32) but lacking direct verification in the isolated vessel (30). Also potentially relevant to vasomotor control was the upregulation of genes for AA metabolism via 12S-lipoxygenase (DA) and CYP450-based monooxygenase (DA, Ao) pathways. The actual role being played by 12S-lipoxygenase, counting among its products the biologically active hepoxilins (25, 27), remains to be ascertained. Equally undefined is the significance of Gal in the neonatal DA, although selective appearance of this transcript may imply the operation of a novel neurohumoral mechanism. Outwardly incongruent with this spectrum of changes is the downregulation in DA of genes for contractile proteins, particularly considering that the same genes are upregulated in Ao. A plausible explanation for this finding is that, at 3 h after birth, DA is in a transition state characterized by a passage from functional to permanent closure. Indeed, there were coincidentally changes in a host of genes—structural, signaling, growth and proliferation linked, and immune system related—that denote a remodeling process being orchestrated or already under way. Characteristic of this process was the concomitance of hyperplastic with proapoptotic influences, confirming earlier data in other species including humans (16, 20, 22, 29). A similar, albeit less prominent, remodeling was present in Ao, with genes for proliferation and apoptosis being in particular not as conspicuous as in DA. In fact, as expected from an organ that is bound to remain viable, there was in the neonatal Ao, but not in DA, a coordinated upregulation of genes encoding a broad range of factors for glucose and lipid metabolism. Furthermore, there was in the same vessel upregulation of adipocyte markers as well as a concerted adjustment of uncoupling proteins, including Ucp1, which is conventionally identified with brown adipose tissue (21). The significance of the latter findings remains unsettled. However, enhanced adipocyte function in Ao may be important for the proper development of the muscle layer (2). A fine regulation of energy homeostasis, on the other hand, may be part of a wider process ensuring independent thermoregulation in the neonate. In the same vein, an adaptive value may be assigned to the birth-related upregulation of genes in both vessels responding to oxidative stress and serving, accordingly, as immediate defense against the transitional rise in blood oxygen tension.

The vessel- and age-linked expression of certain genes (see Table 1) accords with the observed pattern of transitional changes. Specifically, in the fetus both DA and Ao preferentially presented genes encoding structural elements. After birth, however, there was a differential enrichment, with DA expressing genes for vasomotor control and tissue remodeling and Ao expressing primarily genes for metabolic functions and the control of energy expenditure. Collectively, these data reassert a basic dichotomy in the functional arrangement of the two vessels through the adaptation to postnatal life.

A critical question is whether postnatal changes in the gene expression profile of DA reflect a preset programming sequence being initiated by birth or rather result, at least in part, from certain intervening stimuli. Foremost among such stimuli, for timing and impact, would be oxygen, and this possibility was specifically addressed in our investigation. However, in comparing the gene pattern of the hyperoxic fetus with that of the newborn, it turned out that some key features of the naturally closing DA are missing in the vessel being exposed to oxygen alone. Specifically, many genes for tissue remodeling, whether encoding structural or signaling elements, were downregulated by oxygen and upregulated by birth. A similar divergence was noted with allied genes pertaining to cell proliferation and apoptosis. None of the growth-related genes, on the other hand, was upregulated by oxygen. Conversely, genes potentially conditioning the contractile function of ET-1, through upregulation (Gata2) or downregulation (Ednrb), showed coinciding responses in the two situations. In brief, from these data it is reasonable to conclude that the early phase of DA closure, being functional in character, is oxygen linked, whereas the secondary phase, leading to permanent closure, involves a different set of factors conceivably with their own intrinsic programming. Recent evidence (19) implicating ET-1 in the initial component of the oxygen contraction accords with our conclusion. However, oxygen itself, although not contributing directly to the obliteration of the vessel, causes downregulation of genes encoding structural components of cell architecture and the contractile apparatus (see Table 2).

The nature of the stimulus, or stimuli, for DA remodeling and the ensuing permanent closure needs to be settled. Although the operation of a defined genetic program in the natural history of the vessel is beyond doubt (3, 30), secondary events being triggered by the forceful contraction at birth are also expected. Clyman and associates (8, 16) have ascribed importance to the local hypoxia occurring across the wall of the closing DA. Indeed, in accord with this concept, we have found evidence of HIF-1 upregulation in the neonatal DA, standing in contrast with the downregulation of the same gene in the neonatal Ao. Alternatively, or concomitantly, there may be an impact on DA of shear stress resulting from the turbulent flow along a narrowing lumen. Consistent with the latter possibility is the upregulation of genes, whether structural (Itga, VE-cadherin, Cav) or signaling (Pecam1, Klf4) in character, that are eminently susceptible to mechanical stimuli (14, 18, 23, 26, 31). Also significant here is the reported upregulation of the ANG II type 1 receptor on mechanical stress (38). Hence, a synergistic process may ensue, with ANG II expressing its action not only through vasomotion but also as a remodeling agent (5).

Our collective findings, together with existing data, allow us to formulate a novel and comprehensive scheme for the sequence of events taking place in the closing DA (Table 3). The initial phase of functional closure sees the synergistic interaction of contractile (ET-1, Kv channel, ANG II) and relaxant (PGE2) influences being increased and decreased, respectively. ET-1 is assigned a key role because it precedes Kv changes in the sequence of events (19) and, unlike the other putative effectors, is linked in its function with the postnatal rise in oxygen tension. ANG II, on the other hand, may exert a constrictor effect directly and through its known ability to promote ET-1 formation. The subsequent phase of permanent closure has no causal connection with oxygen, which, by itself, may only downregulate the contractile apparatus. Other events come into play to initiate the remodeling sequence, and they are identified with the shear stress on the luminal surface of the closing DA and with the intramural hypoxia resulting from such constriction. Conceivably, these two processes overlap in a mutually potentiating fashion. By extension, one can envision the situation in utero when the DA is exposed to increased oxygen tension. The resulting constriction may in that case involve only ET-1 and the Kv channel. ANG II is, as expected, not affected, and PGE2 action is, if anything, enhanced because of the downregulation of Pde4b. Furthermore, there is no provision for secondary permanent closure because most elements in the remodeling cascade, including its putative mechanosensitive trigger, are downregulated and, moreover, the intramural hypoxia is seemingly unable to promote Hif.


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  		Table 3. Mechanisms for postnatal closure of DA

 
Our study has conceptual and practical implications. Modifications in the expression of structural DA genes under the influence of oxygen are a novel finding with potential relevance to the management of sick infants requiring PGE1 to keep their DA patent. It may account, at least in part, for the lesser effectiveness of the compound in reopening the duct in the normally oxygenated vs. cyanotic infant (15). Likewise, it may explain difficulties being encountered when PGE1 is used over an extended period of time. These patients become progressively less responsive to treatment. This phenomenon has been ascribed to a postnatal change in receptor function (4), but structural alterations being induced by oxygen could be an added factor. Indeed, the complete success of any pharmacological manipulation, including the use of cyclooxygenase inhibitors in the prematurely born infant with persistent DA, may be conditioned by the state of the contractile apparatus. On the other hand, the necessity of a finely orchestrated mechanism for DA closure, being operational postnatally but not antenatally, may explain why permanent DA closure in utero is an exceedingly rare event despite the frequent exposure of the fetus to constrictor drugs (12). Finally, knowledge of the peculiarities of the gene expression profile in the developing Ao may provide a better insight into the impact of fetal and perinatal events as a risk factor for vascular diseases of the adult.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant of the Italian Ministry of Education and Research (MIUR) to F. Coceani. M. Maffei is an Assistant Telethon Scientist. C. Chiellini and E. Grasso are recipients of a Telethon fellowship and a graduate studentship of the Scuola Superiore S. Anna, respectively.


   ACKNOWLEDGMENTS
 
We thank Dr. J. M. Friedman (Rockefeller University) for support with the microarray analysis.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: F. Coceani, Scuola Superiore S. Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy (e-mail: coceani{at}sssup.it).

* M. Costa and S. Barogi contributed equally to this work. Back

1 The Supplemental Material for this article (Supplemental Tables 1–4) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00231.2005/DC1. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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