Main

Neurological impairments such as cerebrovascular accidents and transient ischemic attacks are far too common and increase in prevalence with aging (1). Moreover, in newborn infants hemorrhage into the germinal matrix and periventricular region occurs in 2 to 5 per 1,000 live births and is associated with the development of severe neurological sequelae such as cerebral palsy, convulsive disorders, and other diseases (2). Among very preterm low birth weight (<32 wk gestation; ≤1,500 g) and particularly among extremely low birth weight (<28 wk gestation; <1000 g) infants, the prevalence of brain damage is particularly high (3). These conditions underscore the importance of a well-regulated cerebral blood flow (CBF) during perinatal development. Moreover, the physiologic and biochemical transitions that occur at the time of birth constitute the single most dramatic series of events in the life of an individual. In these few moments of parturition, the central circulatory pattern must change from one based on placental transfer of respiratory gases to one based on pulmonary ventilation. Systemic vascular resistance increases dramatically, as does arterial blood pressure, while pulmonary vascular resistance and pressure fall. Cardiac (i.e., left-ventricular) output initially increases and then slowly decreases over succeeding days. Despite these dramatic changes in cardiac function and vascular resistance, blood flow to the brain increases only slightly to maintain optimal cerebral oxygenation and metabolism (4). In addition to the cerebral vasculature, per se, carotid arteries (CAs) play a crucial role in maintaining optimal CBF (5). Studies have demonstrated a significant pressure gradient from CAs to cerebral arteries (6), probably to minimize the exposure of high pressure to delicate cerebral arteries, and underscore the importance of CAs in the regulation of CBF. Of note, studies suggest that much of the change in systemic pressure results in dilation/contraction of the large arteries that supply the brain (7). Therefore, failure of CAs to effectively regulate the pressure of the blood reaching delicate cerebral arteries may result in their hemorrhagic rupture. Yet other evidence in premature infants suggests that larger arteries are not able to regulate CBF effectively as in near-term babies (5).

In general, in an infant born at 37 wk gestation or thereafter, the cerebrovascular physiologic transitions usually occur in a well orchestrated fashion. At younger ages (<37 wk, preterm; <28 wk, extremely preterm), however, they may not occur properly, with resultant CBF dysregulation (8). In addition to the changes in cardiovascular dynamics, birth also is associated with a large number of major changes in circulating concentrations of a number of vasoactive hormones and metabolites (9). These include marked increases in norepinephrine and epinephrine, cortisol, the prostaglandins (PGF2α, PGI2, and PGD2), angiotensin II, thyroid-stimulating hormone, and triiodothyronine, as well as increases in bradykinin, free fatty acids, and glycerol. In contrast, the concentrations of circulating adenosine, growth hormone, and PGE2 decrease dramatically (9). Each of these compounds plays an important role in the regulation of vascular reactivity, as well as circulation to the brain and other organs.

Of importance, the cerebral vasculature also undergoes a number of changes with maturational development. During the past several decades, the studies of others and our studies have revealed important aspects in the fundamental signaling mechanisms that regulate cerebrovascular contractility with maturational development in the fetus and newborn, as compared with the adult (10,11). Nonetheless, the fundamental, biochemical, and molecular mechanisms responsible for these developmental changes are poorly understood. Some of the most important mechanistic differences include unique features of calcium (Ca2+)-dependent receptor–second messenger coupling with plasma membrane potassium (K+)- and Ca2+-channels, and the virtual dependence of the immature organism on extracellular Ca2+ (as opposed to intracellular Ca2+ stores in the adult) for Ca2+-dependent thick (myosin) filament regulation (12). In addition, many elements of the non-Ca2+-dependent pathway of protein kinase C to specific enzymes such as extracellular regulated kinases (ERK1/2) and their downstream effectors differ in the fetus, as compared with the adult (13). Taken together, these and differences in other enzymes and kinases account for the significantly greater Ca2+ sensitivity of the cerebrovascular contractile mechanisms of the fetus and newborn, as compared with the adult. These studies also emphasize the need to understand the molecular basis of these changes. Unfortunately, and of critical importance, our current understanding of the role of gene expression that underlies cerebrovascular homeostatic mechanisms during maturational development is extremely limited.

To address this vital issue, by use of oligonucleotide microarrays and signal pathway analysis, we tested the hypothesis that the profound age-related differences in the cerebral artery reactivity are associated with significant underlying changes in the gene expression. We examined changes in gene expression in the CAs from four age groups of sheep: premature fetus, near-term fetus, newborn, and adult.

Results

Our results demonstrate profound changes in ovine carotid artery gene expression profiles with developmental maturation from premature fetus to mature fetus, newborn, and adult. Table 1 enumerates the number of genes with up- and downregulated expression (both >two- and >fourfold-change and P value) in premature fetus, near-term fetus, and newborn lamb as compared with adult sheep. In a striking manner, the changes in CA gene expression profiles from premature fetus and newborn lamb, as compared with adult, differed to a similar extent. As compared with those of adults, in the near-term fetal CAs fewer genes showed differential regulation. A similar pattern of increased changes in CA gene expression in premature fetus and newborn lamb with far fewer changes in near-term fetus also was observed in several functional ( Figure 1 ) as well as canonical ( Figure 2 ) gene pathways/networks. Striking is the “U” shaped pattern of these gene expression responses. The main canonical pathways altered in early life (premature fetus, near-term fetus, and newborn) as compared with adult were cell-cycle G2/M DNA damage checkpoint regulation, mitotic roles of polo-like kinases, and cyclins and cell-cycle regulation pathways. Of relevance, these pathways regulate a number of aspects of cellular growth, proliferation, assembly, DNA replication, cell development, maintenance, and so forth. To validate the relative protein expression as development proceeds, Figure 3 demonstrates that the CA expression of the proteins stathmin 1, filamin A (FLNA), and myosin light chain kinase in premature fetus, near-term fetus, and newborn follows the trend of the microarray analysis.

Table 1 Number of genes altered with development
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Figure 1
figure 1

Functional pathways altered with development. Bar graph demonstrates functional pathways altered with development. n was 4 in each experimental group, and all groups were significantly different as compared with adult (P < 0.05). White, black, and gray bars show comparison of adult with premature fetus, near-term fetus, and newborn, respectively.

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Figure 2
figure 2

Chief canonical pathways altered with development. Significant differences (−log(P value)) in the (a) extracellular regulated kinases–mitogen-activated kinase, (b) insulin-like growth factor 1, (c) Ras homolog gene family member A, (d) integrin, (e) role of checkpoint proteins, and (f) actin cytoskeleton signaling pathways in the carotid arteries from premature fetus, near-term fetus, and newborn lamb, as compared with those from adult are shown in a line-graph format. n was 4 in each experimental group, and all groups were significantly different as compared with adult (P < 0.05).

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Figure 3
figure 3

Real-time PCR validation of microarray analysis. Figure demonstrates changes in the expression of (a) staminin 1, (b) filamin A, and (c) myosin light chain kinase mRNA levels in the carotid arteries from premature fetus, near-term fetus, and newborn lamb, as compared with those from adult as determined by quantitative real-time PCR analysis. n was 4 in each experimental group, and all groups were significantly different as compared with adult (P < 0.05).

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Tables 2 , 3 , 4 enumerate the top 20 genes with upregulated expression in CAs from premature fetus ( Table 2 ), near-term fetus ( Table 3 ), and newborn lamb ( Table 4 ). Tables 5 , 6 , 7 list the top 10–20 genes downregulated in CAs from premature fetus ( Table 5 ), near-term fetus ( Table 6 ), and newborn lamb ( Table 7 ), as compared with adult sheep. Table 8 lists the major genes involved in cellular growth, proliferation, and assembly pathways that were significantly upregulated during early life. Similarly, Table 9 lists those pathway genes downregulated in early life, as compared with adult. The downregulated genes belonged to the integrin, actin cytoskeleton, and protein kinase C-Rho Kinase-mitogen activated protein kinase (MAPK) pathways.

Table 3 Top 20 upregulated genes in near-term fetus as compared with adult
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Table 4 Top 20 upregulated genes in newborn as compared with adult
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Table 2 Top 20 upregulated genes in premature fetus as compared with adult
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Table 5 Top downregulated genes in premature fetus as compared with adult
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Table 6 Top downregulated genes in near-term fetus as compared with adult
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Table 7 Top downregulated genes in newborn lamb as compared with adult
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Table 8 Cellular proliferation, growth, and assembly pathway upregulated in early life
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Table 9 Top three downregulated pathways
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Discussion

In comparison with those genes in the adult, this study demonstrates the important changes in gene regulation in ovine CAs with developmental maturation from preterm fetus to term fetus and newborn. The changes in gene expression profiles (both up- and downregulation) follow a U-shaped pattern from preterm fetus to near-term fetus and newborn. The deviations of gene expression (either up- or downregulation) are much greater in premature fetus and newborn lamb than in the near-term fetus. We are not aware of any such report demonstrating that the changes in gene expression profiles are less in a near-term fetus than those in the premature fetus or newborn. These findings underscore the immense changes in gene expression that occur during the perinatal period, birth, and newborn life and how these differ in unique and unexpected manners. Of critical importance, several parameters associated with cerebral blood flow such as PCO2, hemoglobin grams percent, and heart rate, follow a similar U-shaped pattern during preterm, near-term, and newborn life (14). Therefore, perhaps it should be no surprise that the gene expression follows a somewhat similar pattern. Of critical importance, at present the meaning of these findings in a deep sense is not clear. Furthermore, similar to our finding of a U-shaped curve of gene expression during early life, a number of other significant events, such as complete de-methylation and re-methylation of genome, transcriptional silencing of ovum, and histones acetylation of spermatic DNA still require explanation and understanding, and these findings add to this list.

This study demonstrates that collagen triple helix repeat–containing protein 1 (Cthrc1) was upregulated ~450-fold in premature fetus ( Table 3 ); however, its expression decreased 47-fold in near-term fetus and 35-fold in newborn cerebral arteries. Cthrc1 is a gene product with novel biochemical activities, and its ability to reduce collagen deposition by inhibition of Smad2/3 activation plays a major role in vascular development, repair, and fibrosis (15). Of importance, Cthrc1 also increases cellular migration and reduces collagen deposition (16).

Recently, we reported that from an ultrastructural standpoint cerebral arteries of the premature fetus are significantly more fragile than those of the near-term fetus (17). Cthrc1 upregulation may be responsible for reduced collagen contents and therefore increased fragility, leading to higher propensity of the premature fetus for germinal matrix hemorrhage and other intracerebral bleeds; however, further investigation is needed to examine the role of Cthrc1 during fetal life. Similarly, extracellular superoxide dismutase (SOD) expression was significantly downregulated during early life, as compared with adult. Vascular tissue expresses three distinct isoforms of SOD: cytosolic or copper-zinc SOD (CuZn-SOD; SOD1), manganese SOD (Mn-SOD) localized in mitochondria (mitochondrial SOD or SOD2), and an extracellular form of CuZn-SOD (EC-SOD; SOD3) (18). Because no selective pharmacological inhibitors of individual SOD isoforms are available, the functional importance of the different SODs has been difficult to define. However, our finding of significantly downregulated expression of specifically EC-SOD3 suggests a distinct role in early life. SOD plays a crucial role in conversion of superoxide anion (O2–) to H2O2; which is further converted to H2O by the actions of glutathione peroxidases and peroxiredoxins. Also of importance, in this study we observed that along with reduced expression of SOD3, there was a significantly increased expression of both glutathione peroxidase (GPX8) and peroxiredoxin4 (PRDX4). Therefore, the system is geared toward reduced production and rapid clearance of H2O2. At present, the clear rationale of this is not known.

Cell Proliferation, Growth, and Assembly Pathway Gene Expression Is Upregulated During Early Life

To no surprise, many genes involved in cell-cycle regulation, DNA replication, chromosome assembly, and other components of CA cell replication, growth, and assembly demonstrated significantly increased expression. However, of importance, the fold changes of these genes were significantly greater in the premature fetus and newborn than in the near-term fetus. This emphasizes the complexity of the changes that occur at the several developmental ages. This study also demonstrates that aurora kinase A and B, several cyclins, centromere proteins, and replication factors are significantly upregulated during early life. Overexpression of Aurora A and Aurora B can lead to genetic instability (gain or loss of whole chromosomes) by the overphosphorylation of normal cell-cycle targets and the aberrant phosphorylation of cytoplasmic targets (19). The resultant chromosomal instability is a common feature of many cancer types (20). A significant over expression of these kinases during fetal life, however, is in contrast to these findings, and suggests that the precise role during early life requires further investigation.

Moreover, ubiquitin-conjugating enzyme e2c (UBE2C) was upregulated almost 260-fold in the premature fetus and its expression fell to ~66-fold in the near-term fetus. Nonetheless, this indicates high expression of this gene during early life. Of note, excessive UBE2C is known to disrupt normal chromosome segregation or even lead to mis-separation of the chromosomes (21) and may lead to malignancies (22). However, the significance of such high levels in early life is unknown.

MAPK-ERK Signaling Pathway Gene Expression Is Downregulated in Early Life

The MAPK pathway is a major signaling cascade involved in cellular growth and development. In this study, several components of the MAPK pathway, including MAPK3, MAP2K2, and MAP kinase interacting serine/threonine kinase 1 and 2, were expressed several fold lower during early life. Of importance, we also observed a significant reduction in the expression of protein kinase C – delta in fetus and newborn, as compared with adult. Recent studies demonstrate that upstream to the MAPK pathway, protein kinase C – delta is necessary for the activation of MAPK3 (23). Thus, the findings suggest that several components of the MAPK-ERK pathway are suppressed during early life. Of note, evidence supports the idea that downregulation of this pathway is advantageous for organism survival and well-being. For instance, MAPK3 downregulation has been demonstrated to be beneficial for striatum-dependent long-term memory (24), reduced adiposity, and protection from high-fat diet–induced obesity and insulin resistance (25). Similar results of MAPK-mediated negative regulation of self-renewal cell division have been demonstrated in developing plant cells (26).

On the basis of this study and the above-mentioned reports, reduced expression and down-regulation of MAPK3 would appear to play a critical role in development. In previous reports, we have demonstrated that MAPK plays a significantly reduced role in cerebral arterial contractility during fetal life, compared to that in the adult (27). Nonetheless, the MAPK cascade involvement in negative regulation of vascular growth and development and its role in myofilament Ca2+ sensitivity necessitates further investigation.

Actin Cytoskeleton Pathway Gene Expression Is Downregulated in Early Life

In CAs several important members of the actin cytoskeleton canonical pathways were demonstrated to have reduced expression during early life, as compared with the adult. Of note, FLNA expression was downregulated to a significantly greater extent in premature than in near-term fetal arteries. The reduced expression of FLNA also was significantly more pronounced on real-time PCR analysis, as compared with that of microarray analysis. Several explanations could account for this. For instance, microarray examination is based on the ratio of the densitometric analysis of the signal, whereas in PCR the florescence signal is exponentially weighted to the power of 2 to correct for the doubling with each cycle. This makes the PCR technique much more sensitive as compared with the microarray analysis. Nonetheless, with both techniques, the finding that there is a significantly greater reduction of FLNA in the premature vessels remains the same. Studies have demonstrated that filamin exists in three isoforms, FLNA, filamin B (FLNB), and filamin C (FLNC) (28). Of critical importance, FLNC has been shown to have a restricted expression in skeletal and cardiac muscle (28) and FLNA and FLNB play a crucial role in corticogenesis and brain development (29). However, our study shows significantly greater reduction of FLNA expression in premature CAs, as compared with those of the near-term fetus. Currently, a clear rationale of such a finding is unknown. Nonetheless, this study suggests an important role of FLNA in vascular development. Our results demonstrate further that formin binding protein 1 was reduced significantly during early life. Formin binding protein 1 has been implicated in smooth muscle phenotype switching to a contractile type, and this agrees with our findings (30). Moreover, the actin cytoskeleton signaling cascade has been implicated in a host of functions, including cell motility, surface remodeling, cell shape changes during mitosis, muscle contraction, separation of daughter cells by the contractile ring during cytokinesis, cell–cell, and cell–substrate interactions together with adhesion molecules, transmembrane signaling, endocytosis, and secretion (31). Downregulation of this pathway in CAs may suggest that it plays a critical role in the above-mentioned aspects of smooth muscle contractile and/or development pathway and/or phenotype.

Integrin-Signaling Pathway Gene Expression Is Downregulated During Early Life

Another important signaling pathway altered with development was that of integrin signaling ( Table 8 ). This study demonstrates that major components of the integrin-signaling cascade expression were downregulated in both immature and near-term fetus, as well as newborn CAs. Integrins have been implicated in several fundamental cellular functions such as cellular movement, stabilization, and adhesion (32), as well as internal cellular cytoskeleton organization (33). Evidence also supports the idea that integrins mediate intracellular signaling through janus kinase/signal transducer and activator of transcription and MAPK pathways (34). Not only integrins but also their downstream mediators, such as those involved in MAPK pathway expression, were downregulated. In contrast, caveolin, a scaffolding protein in the integrin signaling cascade, was expressed to a significantly greater extent in premature fetus and newborn, whereas its expression was reduced in near-term fetus. Of note, caveolin links integrin subunits to the tyrosine kinase Fyn, an initiating step in coupling integrins to the Ras-ERK pathway and promoting cell-cycle progression (35). Moreover, it is a negative regulator of the Ras-MAPK cascade (36). As noted, MAPK pathway expression is downregulated in early fetal life, and caveolin, a negative regulator of this pathway expression is upregulated. This suggests an active downregulation of the MAPK cascade and further indicates that MAPK inhibition plays a critical developmental role during early life.

Perspective

Overall, results of this study demonstrate significant alterations of gene expression with maturation of the cranial vasculature. For the first time, we demonstrate that, as compared with more active gene expression of several signaling pathways in the premature fetus and newborn organism, in the near-term fetus differential gene expression is attenuated significantly. These findings agree with the concept of molecular mechanisms acting in an integrated manner to regulate both phenotypic and mechanical plasticity in vascular smooth muscle cells (37), and may be an important step for the preparation of the fetus for birth. Of importance, our study raises a number of questions regarding the regulation of changes in vascular gene expression and their biological significance in the developing premature fetus, near-term fetus, and newborn. This study also provides a basis for future studies to explore the importance of changes in the major signal transduction pathways during early vascular development and the function of these changes. Perhaps most important, this study suggests avenues in which to target the developing cerebral vasculature for gene therapy to ameliorate the serious pathophysiologic disruptions that may occur during early life (38).

Methods

Experimental Animals and Tissues

All experimental procedures were performed within the regulations of the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, “The Guiding Principles in the Care and Use of Animals” approved by the Council of the American Physiological Society, and the Animal Care and Use Committee of Loma Linda University. For these studies, we used CAs from premature (95-gestational-day) fetus, near-term (~140-gestational-day) fetus, newborn lamb (1–5 d old), and nonpregnant adult sheep (18–24 mo) obtained from Nebeker Ranch (Lancaster, CA). For each experiment four animals were used; in case of fetal twins only one of the twins was included in the study.

Pregnant and non-pregnant ewes were anesthetized with thiopental sodium (10 mg/kg, i.v.), and anesthesia was maintained with inhalation of 1% isoflurane in oxygen throughout surgery. Following delivery of the fetus by hysterotomy, the fetuses and ewes were euthanized with an overdose of the proprietary euthanasia solution, Euthasol (pentobarbital sodium 100 mg/kg and phenytoin sodium 10 mg/kg; Virbac, Ft. Worth, TX). Studies were performed in isolated CAs cleaned of adipose and connective tissue. To avoid the complications of endothelial-mediated effects, we removed the endothelium by carefully inserting a small wire three times, as previously described (11).

Tissue Collection and Microarray Processing

In previous studies, we have described this technique in detail (39). Microarray analysis was conducted by using commercial services of GenUs Biosystems, Northbrook, IL. Briefly, tissue samples were lysed in Tri-reagent (Ambion, Austin, TX) and total RNA was isolated using phenol/chloroform extraction followed by purification over spin columns (Ambion). The concentration and purity of total RNA was measured by spectrophotometry at optical density 260/280 and the quality of the total RNA sample was assessed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies, Santa Clara, CA).

Labeled complementary RNA was prepared by linear amplification of the Poly(A) + RNA population within the total RNA sample. Briefly, <1 µg of total RNA was reverse-transcribed after priming with a DNA oligonucleotide containing the T7 RNA polymerase promoter 5′ to a d(T)24 sequence. After second-strand complementary DNA synthesis and purification of double-stranded complementary DNA, in vitro transcription was performed using T7 RNA polymerase. The quantity and quality of the labeled complementary RNA was assayed by spectrophotometry and the Agilent Bioanalyzer.

One µg of purified complementary RNA was fragmented to uniform size and applied to Agilent Sheep Gene Expression Microarray, 8 × 15K (Design ID 019921, Agilent Technologies) in hybridization buffer. Arrays were hybridized at 65 °C for 17 h. in a shaking incubator and washed at 37 °C for 1 min. Rinsed and dried arrays were scanned with an Agilent G2565 Microarray Scanner (Agilent Technologies) at 5 µm resolution. Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies). Annotations are based on the Agilent eArray annotation file dated January 2010.

Pathway/Network Analysis

Each gene was annotated manually using NCBI Blast Search, Unigene, Entrez, or other databases. We then analyzed the annotated genes using the Ingenuity Pathway Analysis Program (Ingenuity Systems, Redwood City, CA).

Real-Time PCR Validation

To validate the results of the microarray analysis, we chose stathmin 1, FLNA, and myosin light chain kinase genes that were highly regulated by development during early life, as compared with adult life, for analysis using real-time PCR. Using the same probe sequences as those on the microarray chip, we designed primers with the use of Primer 3 web-based software (http://frodo.wi.mit.edu/primer3/). The primers were synthesized by Integrated DNA technologies (Coralville, CA). Total RNA (1 µg per reaction) was reverse transcribed using Quantitect reverse transcriptase kit (Qiagen, Valencia, CA). Relative expression was normalized to 18S RNA and fold changes were calculated using the ΔΔ cycle threshold (CT) method (40). Samples were analyzed on the Roche LightCycler 1.5 (Roche, Indianapolis, IN).

Statistics

To compare individual expression values across arrays, raw intensity data from each gene were normalized to the 75th percentile intensity of each array. Only genes with values greater than background intensity for all samples within each group were used for further analysis. Differentially expressed genes were identified by twofold change and Welch t-test P values <0.05 between each treatment group and its age-specific normoxic control. Statistical significance in the real-time PCR data was determined by one-way ANOVA and post-hoc Newmans–Keul test.

Statement of Financial Support

The studies were supported by United States Public Health Service grant HD-03807 (to L.D.L.).