INTRODUCTION

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DURING THE MATURATION OF PHYSIOLOGICAL SYSTEMS, many of the mechanisms governing various functional outcomes are altered. With increasing age, these alterations may in turn lead to functional impairments and exacerbate disease. The vascular system has been shown to follow this path with regard to a variety of functions, including blood vessel reactivity (20, 35) and vascular growth and remodeling (3, 24, 27). Studies from our laboratory have focused on understanding processes involved in collateral artery remodeling after chronic elevation of blood flow. Currently, little is known about how maturation and aging affect the capacity for collateral development. The clinical observation/experience is that collateral growth (32) and flow-induced luminal enlargement of arteries (8) occur in adult humans. However, flow-mediated remodeling in large conduit arteries, which has many mechanistic similarities to collateral/resistance artery remodeling, has been observed to be impaired with maturation (3, 24).

Arterial remodeling occurs after chronic elevation of blood flow and is a complex process involving flow/shear stimulus transduction and endothelial activation (5, 26). After transduction and activation, remodeling of the entire arterial wall is accomplished through changes in protein expression/activity and cell growth. Luminal dimensions are altered to normalize wall shear stress (3, 24, 40, 46). Age-dependent alterations in any of the mechanisms involved in flow-induced arterial remodeling could influence the capacity for luminal adjustment in response to altered shear. The primary purpose of the current study was to determine whether flow-induced expansion of resistance arteries forming collateral pathways is impaired with maturation and to provide insight regarding where specific abnormalities might occur if the remodeling capacity was suppressed. Intimal and medial cell growth were evaluated with morphometric analysis. Regulation of endothelial nitric oxide (NO) synthase (eNOS) mRNA was evaluated with RT-PCR. Proteomic analysis was performed to determine whether the pattern of protein expression was altered differently in collateral vessels of mature and young rats.

	METHODS

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Experimental design. The Indiana University-Purdue University at Indianapolis Institutional Animal Care and Use Committee approved all procedures performed in this study. After experimentation, animals were euthanized with an overdose of anesthetic and aortic transection. A collateral artery model (41) was created within young and mature male Wistar rats (Harlan; Indianapolis, IN) weighing ~200 g (~2 mo old) and ~600 g (>8 mo old), respectively. Acute animal studies were completed with both groups for determination of blood flow and shear rate. Chronic experiments were performed to evaluate luminal expansion and wall remodeling after model creation. The majority of these experiments were terminated at 1 wk because the most rapid phase of luminal expansion occurs during this time, when diameters in young animals are increased ~35% (38, 40). A few mature animals were also studied after 4 wk to determine whether luminal expansion might simply be delayed in mature animals. Additional chronic experiments were used to evaluate mRNA levels and protein expression at 1 and 2 days, respectively, after model creation. Significant alteration in gene expression has occurred in collateral or shear-loaded vessels of young animals by this time (38).

Acute studies. After rats were anesthetized (100 mg/kg ip thiopental), a tracheostomy was performed to maintain a patent airway, and a cannula was inserted into the left carotid artery to inject microspheres and monitor arterial pressure. After laparotomy, the ileal portion of the bowel and the cecum were gently placed into a support chamber attached to the rat's abdomen and bathed in 37°C PBS. A collateral-dependent region of the bowel containing 45-55 first-order arterioles was created by the sequential ligation of 3-4 ileal arteries located near the cecum (41). This selectively increases flow in the ileal arteries immediately adjacent to the collateral-dependent region. Tissue blood flow was measured with the use of standard fluorescent microsphere techniques described for rats as previously reported (40). After model creation, a reference blood sample was started that allowed blood to freely flow from a femoral cannula (polyethylene-10) into a preweighed 15-ml conical tube. Ten seconds after the reference blood sample was started, microspheres (0.1 ml of 10 µm, 3.3 × 10⁶/ml red fluorescent; Molecular Probes) were injected through the carotid cannula, followed by a 0.3-ml flush (physiological Ringer solution). Approximately 0.5 ml of blood was obtained during the 90-s collection period. Resting arterial diameters were then measured with videomicroscopic techniques.

Chronic studies. In brief, the animals were anesthetized with sodium pentobarbital (50 mg/kg im) and administered atropine (0.4 mg/kg im) to prevent airway congestion. The vascular model was created as described for the acute studies. Once the ligations were completed, the bowel segment was maximally dilated by the addition of a dilator cocktail (10⁴ M adenosine and 10⁵ M sodium nitroprusside) to the suffusion solution. Video images of the experimental (high flow) arteries and adjacent control arteries were recorded for later diameter measurement (Olympus SZH dissecting microscope, Olympus; Hamamatsu model C2400-50 charge-coupled device videocamera, Hamamatsu Photonics; Sony SVO-9500MD SVHS VCR and Sony Trinitron monitor model PVM-1343MD, Sony Medical Systems; total magnification  ×50). The bowel was carefully placed back into the peritoneal cavity, and the incision was closed in two layers with running sutures (3-0 Dexon, Davis & Geck). The rats were administered antibiotics for 3 days postoperatively (1.1% tetracycline in drinking water) and allowed free access to food and water. At the final observation, laparotomy and measurement of maximally dilated diameters were repeated.

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5

Analysis of eNOS mRNA: relative semiquantitative RT-PCR. Multiplex relative quantitative RT-PCR was used to measure differences in eNOS mRNA expression between control and collateral arteries in young and mature rats. Total RNA was isolated (RNeasy Purification Kit; Qiagen; Valencia, CA) from arteries treated with RNA preservation solution (RNAlater, Ambion; Austin, TX). The arterial tissue was homogenized [FastPrep Instrument, BIO 101 (Savant); Vista, CA] and the homogenate was digested with proteinase K before RNA purification (Qiagen protocol). Before reverse transcription, total RNA was treated with DNase to remove genomic DNA contamination (DNA-free, Ambion). Reverse transcription was performed with random decamer priming using 400 ng total RNA (Ready-To-Go You-Prime First-Strand Beads, Amersham Pharmacia Biotech; Piscataway, NJ). Primers for eNOS were synthesized from a previously published sequence (44), and thermal cycling conditions were 94°C, 57°C, and 72°C, 30 s each, for 35 cycles (Qiagen Taq DNA Polymerase). Pilot experiments were conducted to ensure that PCR reactions were terminated in an exponential range. To ensure that the invariant endogenous control (18S rRNA) was amplified with the same efficiency as eNOS, Competimer technology (QuantumRNA 18S Internal Standards, Ambion) was used. PCR products were separated by electrophoresis on 2% E-Gels (Invitrogen; Carlsbad, CA) and bands were visualized and digitally captured with an ImageMaster VDS (Amersham Pharmacia Biotech). Densitometric analysis was performed utilizing Intelligent Quantifier software (BioImage; Ann Arbor, MI).

Two-dimensional electrophoresis and peptide mass fingerprinting. Protein expression was evaluated by two-dimensional (2-D) electrophoresis and peptide mass fingerprinting. In these studies, the model was created in young and mature animals as described above. Two days after model creation, a laparotomy was performed, and the caudal aorta was perfused with cold (0-4°C) PBS to remove all blood from the ileal arteries. The bowel with intact mesentery and vasculature was excised and placed on a chilled silcone disc in a petri dish and covered with chilled saline. The arteries of interest were dissected from their vascular bundle, placed in microcentrifuge tubes on dry ice, and then stored at 80°C for later analysis. These experiments were completed using a total of six young and six mature rats. To provide adequate protein amounts, control and collateral arteries from each of two animals were pooled into pairs (two control and two experimental arteries per animal), yielding three sets of four control and four collateral arteries of each group (young and mature). The arteries in each tube were solubilized by adding 40 µl lysis buffer containing 9 M urea, 4% Igepal CA-630 ([octylphenoxy]polyethoxyethanol), 1% dithiothreitol, and 2% ampholytes (pH 8-10.5) directly to each sample. The samples were sonicated with a Fisher Sonic Dismembranator using 3× 2-s bursts at instrument setting 3 every 15 min for 1 h, after which the fully solubilized samples were transferred to a cryotube for storage at 80°C until thawed for 2-D electrophoresis analysis.

Data analysis. All data were entered into a spreadsheet. Animal averages for normal and collateral arteries were calculated for statistical comparisons. Two-way ANOVA with two repeated factors was used for comparisons of in vivo data (diameters, arterial flow, and wall shear rate). For the statistical evaluation of the histological and morphometric data, two-way ANOVA was performed, with vessel type (collateral or normal) as a repeated factor within animals. Bonferroni comparisons were used to evaluate significant differences (P < 0.05). All measurements are reported as means ± SE.

	RESULTS

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Blood flow. For equivalent arterial occlusion, blood flow and shear rate were significantly greater in the collateral compared with the control arteries within each group (Table 1). Collateral artery blood flow was greater than control in both the young (120 ± 21%) and mature (144 ± 58%) groups. There was not a significant difference in the percent flow increase between groups. Similarly, the percent change in wall shear rate relative to control was not statistically significant between groups.

Luminal dimensions. Averages of maximally dilated inner diameters of control and collateral arteries in young and mature animals at the time of model creation (day 0) and 1 wk later are depicted in Fig. 1. At day 0, both control and collateral arteries were larger in mature than young animals due to maturational effects (whole body growth). These measurements made on the same arteries at days 0 and 7 indicate that the only increase in inner diameter occurred in young collaterals. The effect of the larger initial diameter in mature rats is eliminated as shown in the presentation of the data in Fig. 2 as the ratio of day 7 to day 0 diameters. As Fig. 2 shows, only the collateral arteries in young rats had a day 7-to-day 0 diameter ratio >1.0. The increase in the collaterals of young animals was 33 ± 7%. Data from additional experiments in mature rats (n = 4) revealed that there was not simply a delay in the luminal expansion of older animals because the luminal diameters of collateral arteries were not significantly enlarged even after 4 wk.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Averaged maximally dilated diameters of control (Con) and collateral arteries (Col) for young and mature rats at the initial (day 0) and final (day 7) times of observation. Measurements were made from 8 young and 8 mature rats in which control and collateral diameters were available at both time points. *P  0.05 vs. control arteries; P  0.05 vs. day 0; P  0.05 vs. young rats.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   To illustrate diameter changes between observation times, arterial diameters measured at 1 wk (T2) were expressed relative to the original diameter at the time of model creation (T1). Significant enlargement occurred only in the collaterals of young animals. *P  0.05 vs. control arteries.

Morphology. The numbers of intimal cell nuclei in arterial cross sections of control and collateral arteries of young and mature rats are shown in Fig. 3. Cross-sectional intimal nuclear number was similar between groups for both vessel types. Relative to control arteries within the same animals, there were 57 ± 10 and 52 ± 14% more intimal nuclei in collaterals of young and mature animals, respectively (P < 0.001).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Means of the animal averages for endothelial cell nuclear number in arterial cross sections of control and collateral arteries of young and mature rats. The greater number in collateral relative to control arteries was similar in both young and mature rats. *P  0.05 vs. control arteries.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Means of the animal averages for medial area in arterial cross sections of control and collateral arteries of young and mature rats. The increase in medial area in collateral relative to control arteries was similar in both young and mature rats. *P  0.05 vs. control arteries.

Semiquantitative eNOS mRNA expression. The representative micrograph in Fig. 5 illustrates the increased expression of eNOS mRNA observed in mature collateral relative to control arteries. 18S rRNA was used an an internal standard. Collateral eNOS mRNA/18S was normalized to the same animal control [(collateral eNOS mRNA/18S)/(control eNOS mRNA/18S)] as an index of increased eNOS mRNA in the collaterals. This index indicated that eNOS mRNA was increased at least as much in mature as young collaterals (5.43 ± 2.17 vs. 1.73 ± 0.35, P = 0.095).

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Representative micrograph illustrating the results of RT-PCR for endothelial nitric oxide synthase (eNOS) mRNA and 18S in control and collateral arteries of mature rats. Animal averages (n = 6) of eNOS mRNA to 18S in young rats were 1.11 ± 0.27 for control and 1.97 ± 0.63 for collateral arteries. In mature animals (n = 5), these averages were 1.31 ± 0.36 for control and 5.59 ± 1.88 for collateral vessels.

Proteomics. Sample proteins from artery homogenates separated by 2-D electrophoresis and analyzed by PDQuest are shown in the reference pattern shown in Fig. 6. This representative pattern illustrates the 1,192 proteins that were detected and matched in the sample gel patterns. A comparison of protein patterns between control arteries of young and mature animals revealed a significant difference (P < 0.05) in the abundance of 28 proteins (21 higher and 7 lower in the young animals). When we compared the protein patterns of control to collateral arteries, 15 arterial proteins differed in abundance (4 higher and 11 lower) in the young animals, whereas 24 (5 higher and 19 lower) proteins had differing abundances in the mature animals. There was no overlap in the proteins displaying altered expression in response to elevated flow (collaterals) between young and mature rats; none of the 15 proteins displaying altered expression in young rats exhibited altered expression in mature animals. Twenty-eight of the proteins in the pattern were excised from the 2-D gels and unambiguously identified by peptide mass fingerprinting (Table 2). Relative abundances of these identified proteins, based on the mean protein abundance in the young control arteries, were calculated and are summarized in Table 3. Three of these identified proteins had decreased expression in mature relative to young animals. The level of annexin V was decreased in both control and collateral arteries of mature rats. Myosin regulatory light chain 2 and heat shock cognate 71 kDA had diminished levels in collaterals of mature relative to young animals. Of the 28 identified proteins, vimentin, smooth muscle actin, heat shock proteins [27 kDa (HSP27) and 70 kDa (HSP70)], and probable protein disulfide isomerase ER-6 had increased expression in the collaterals of young but not mature animals. In collaterals of mature rats, mitochondrial HSP (HSP60), guanine deaminase, and -enolase had increased expression relative to same animal controls.

View larger version (70K): [in this window] [in a new window]   Fig. 6.   PDQuest reference pattern showing the coordinate locations of 14 of 28 identified proteins from ileal artery samples separated by two-dimensional electrophoresis and identified by peptide mass fingerprinting. Bar graphs indicated mean abundance data (n = 3) for each protein. From left to right, the bars represent young controls, young collaterals, mature controls, and mature collaterals.

	DISCUSSION

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Significant luminal expansion of preexisting arteries forming collateral pathways occurred in the young rats of this study. Two initiating stimuli have been proposed for collateral development: ischemia (4, 14, 22) and elevated wall shear (11, 29). Because both ischemia in tissues distal to the occlusion and shear elevation in preexisting collateral pathways may occur concurrently during arterial occlusion, it can be difficult to determine the primary stimulus. Indeed, the primary mechanism responsible for collateral growth remains a controversial issue. Whereas tissue blood flow is not reduced in this model under resting conditions with chyme in the bowel of anesthetized animals (41), it could be compromised in conscious animals, particularly during peak postprandial hyperemia. However, our initial study (41) with this model demonstrated that arteriolar adaptations did not occur at the center of the collateral-dependent pathway, where we expected any ischemic stimuli to be greatest. The mesenteric arteries that form collateral pathways and exhibit luminal expansion in our model are located 3 cm from the center of the collateral-dependent region. Wall shear level is increased at this location (Table 1), and the rate and magnitude of expansion is correlated with the shear stimulus (38). The observations that arterial luminal expansion in this model occurs where wall shear is elevated, is correlated with the degree of shear elevation, and ceases after wall shear rate is normalized (6, 38, 40, 41) suggest that wall shear is important at least as a molding influence if not as the primary stimulus. For these reasons, we consider the luminal expansion observed in these collaterals to be flow or shear mediated. This remodeling would also be expected to be influenced by any alterations in circumferential wall tension or pressure experienced by these vessels or transient episodes of ischemia. Regardless of the initiating or primary stimuli, the current study allows for comparison and contrast between adaptations in young and mature rats for equivalent arterial occlusion and the evaluation of the protein profile associated with collateral development.

In the current study, equivalent arterial occlusion in young and mature rats produced similar elevation of blood flow and wall shear rate within the collateral arteries (Table 1), and similar intimal and medial remodeling events occurred within the arterial walls (Figs. 3-5). Yet luminal expansion occurred only within collaterals of young animals (Figs. 1 and 2). We had anticipated that collateral development in the mature animals might be suppressed but not completely impaired. However, our observation of impaired flow-mediated expansion in resistance arteries forming collateral pathways is consistent with previous studies of large arteries in mature rats and rabbits (3, 24). Brownlee and Langille (3) reported 2 mo after a ~50% increase in common carotid arterial flow that arterial diameter increased 18% in weaning rabbits but was not increased in adult rabbits. For a similar flow increase in the common carotid artery, Miyashiro et al. (24) observed at 1 mo an increase in outer diameter of ~30% in weanling (99 g) and 10% in older (200 g) rats, but luminal area or inner diameter was not increased in the older animals. In these studies with carotid arteries (24), wall shear forces were restored to control levels in the younger but not older animals. Other studies, however, have observed increases in the luminal diameter of basilar arteries in mature rabbits (18) and iliac arteries of monkeys (46) after 4 or more wk of elevated blood flow. While there are a number of potential explanations for these differences between the former (3, 24) and latter (18, 46) studies, it is likely that the results may be related to the initial flow or shear stimulus level. We have previously shown that differences in the initial stimulus level can produce differences in the magnitude and rate of luminal expansion (38) in young (~200 g) rats. Shear-mediated arterial remodeling and collateral development in mature animals may require higher levels of shear alteration than young animals.

We had expected that cell growth responses would be abnormal if flow-mediated collateral development was impaired. Yet our findings in arterial cross sections of nuclear cell numbers in the intima and of medial area indicate similar responses in these wall layers of collaterals in young and mature animals (Figs. 3 and 4). Indeed, an important conclusion of this work is that the absence of luminal expansion does not indicate that wall remodeling has not occurred. The results within the intima may be of special significance. Masuda et al. (21) have shown that changes in endothelial cell numbers and morphology precede flow-dependent vascular enlargement. Vascular endothelial cells are thought to be the primary sensors of shear with the vascular wall. With age, a reduced ability of endothelial cells to sense or transduce a shear stimulus to the cell interior could impair the remodeling process. The observation that endothelial cell nuclei number is increased (Fig. 3) in the collateral arteries of the mature rats suggests that shear elevation was detected by the endothelial cells and that appropriate transcription and translation occurred to produce endothelial cell proliferation. The remodeling within both the intima and media would seem to indicate that major components of the remodeling process are intact in mature animals, even though luminal expansion did not occur.

eNOS has been one of the most studied of the endothelial genes known to be regulated by shear. We have recently shown that its expression in our model also varies with shear level (38). An important role for eNOS and its product, NO, in shear-mediated remodeling and collateral artery development has been suggested by several studies (22, 24, 36-38). We performed quantitative RT-PCR for this molecule as a preliminary test to determine whether an abnormality spanning from signal transduction through posttranscriptional regulation might exist for this molecule in mature vs. young collateral arteries. Comparison of eNOS mRNA/18S for same animal control and collateral vessels between young and mature rats suggests that the increase in collateral eNOS mRNA is not suppressed in mature animals (Fig. 5). Thus it appears that the shear-dependent regulation of eNOS mRNA levels do not contribute to the impaired response of other molecules whose expression is shear sensitive. Additional studies are needed to investigate transcriptional through posttranslational regulation of this and other shear-sensitive molecules during maturation and aging.

Proteomic analyses were performed to investigate the possibility that biochemical abnormalities may be responsible for impairment of flow-mediated remodeling observed with maturation. A recent review of the potential application of proteomics to the cardiovascular system considered that the technique can be used to determine whether specific experimental treatments or pathology alters the protein profile, even without protein identification (2). We performed a protein profile analysis (proteomics) of arteries of young and mature rats 2 days after arterial occlusion. Differential protein expression was clearly observed between control arteries of young and mature animals and between control and collateral arteries of both young and mature animals (Table 3 and RESULTS). About 2% of the proteins (28 of 1,192) observed had altered expression with maturation. Three times as many proteins exhibited downregulation with age than upregulation. Annexin V was one of the identified proteins that exhibited an age-dependent decrease in expression. This is consistent with a previous study that observed levels of annexin V in several tissues of rats to decrease from postnatal to adult life (7). An additional 3% of the proteins observed (39 of 1,192) exhibited altered expression in collateral vs. control arteries; 15 proteins in young rats and 24 proteins in mature rats. More proteins had decreased than increased expression in the collaterals. While the significance of altered expression for specific proteins remains unknown, the altered expression of vimentin and HSPs may be important in the differences observed in collateral remodeling. Vimentin is a component of the endothelial cytoskeleton that appears to be involved in flow-induced dilation and remodeling (9, 30). Endothelial vimentin content has been shown to be greater in vessels exposed to higher flows and pressures (31). Furthermore, vimentin deficient mice exhibit impaired flow-dependent dilation (9) and are reported to have abnormal remodeling in response to altered flow (30).

In the collaterals of this study, expression was increased for several proteins that have not been previously shown to exhibit shear-dependent expression. These proteins include HSP27 and HSP70 in young rats (>100% increase) and in mature animals, -enolase (37% increase) and HSP60 (>50% increase relative to mature control). The exact physiological significance and the mechanism(s) responsible for upregulation in this system remain to be determined. HSPs have antiapoptotic properties (13) and may participate in the stabilization of microfilaments and enhancement of cell growth (for a review, see Ref. 19). HSP27 may be an intermediary in shear-induced signaling and has previously been shown to undergo phosphorylation but not altered expression in cultured endothelial cells exposed to shear (19). Previous studies have shown stress-induced expression of HSP70 in vascular cells to be greatly diminished with age (17, 39). Enolase has some similarities to HSPs (25). It is a protein with multiple functions that can act as a plasminogen receptor on endothelial cells and may be involved in transcriptional regulation (25). It is also one of five hypoxia-associated proteins in endothelial cells (1). The gene for enolase is transcriptionally regulated by hypoxia-inducible factor (HIF)-1 (12). The expression of HSPs is increased in many types of stress, including hypoxia or ischemia (33). The elevation of enolase and HSPs could be interpreted to indicate the presence of an ischemic stimulus in these vessels. However, it is not clear to the authors how hypoxia could be responsible for the induction of these proteins in these collateral arteries because they have elevated blood flow under both resting and maximally dilated conditions (41). HSPs are also known to be upregulated by free radicals, including NO (45) and superoxide (15), and Sandau et al. (28) have recently reviewed and reported evidence that HIF-1 can be activated by growth factors, cytokines, and NO. Production of NO and reactive oxygen species is increased with elevated flow or shear in arteries in vivo (10, 16), and elevation of these free radicals could also explain the upregulation of HSPs and enolase in our model. Additonal studies are needed to investigate the role of these molecules in collateral development and to determine the specific mechanisms by which their expression is regulated, especially the role of shear stress, ischemia/hypoxia, and free radicals.

As observed for larger arteries, shear-mediated luminal expansion is impaired in resistance arteries with maturation. While an unexpected observation of this report was that intimal hyperplasia and medial layer hypertrophy occur to a normal extent in mature resistance arteries exposed to chronically elevated flow, maturation influences protein expression in both control and flow-loaded collateral arteries. Whereas endothelial dysfunction indicated by endothelial-dependent dilation advances with age (23, 34, 42), endothelial proliferation and eNOS expression were not found to be abnormal in the mature collaterals of this work. Future studies are warranted to investigate mechanisms that lead to altered protein expression, the role of specific proteins that display altered expression, and the degree to which altered matrix metalloproteinase activity or collagen cross-linking with maturation contribute to the suppression of collateral development and shear-induced luminal expansion. Identifying the responsible mechanisms should provide insight regarding potential therapies to enhance collateral development.