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1 Departments of Surgery, 2 Anatomy and Cell Biology, and 3 Cellular and Integrative Physiology, Indiana University Medical Center, Indianapolis, Indiana 46202
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The magnitude of shear stimulus has been shown to determine the level of growth factor expression in cell culture. However, little is known regarding what effect shear level has on specific arterial wall remodeling events in vivo. We have hypothesized that the rate of luminal diameter change and specific remodeling events within the arterial wall layers are dependent on shear level. Selective ligations were made to alter the number of microvascular perfusion units of mesenteric arteries within the same animal to ~50%, 200%, and 400% of control. Arterial blood flow and wall shear rate were correlated with the degree of alteration in perfusion units. Luminal diameters were decreased in 50% arteries by day 2 and increased ~17% and 33%, respectively, in 200% and 400% arteries at day 7. The rate of diameter change was greatest in 50% and 400% arteries. Wall areas (medial +37%; intimal +18% at day 2) and cell densities (intimal +26%; adventitial +44% at day 2) were altered only in the 400% arteries. A positive correlation existed by day 2 between endothelial staining for endothelial nitric oxide synthase and shear level. The results demonstrate that shear level influences the rate of luminal expansion, specific remodeling events within each wall layer, and the degree of endothelial gene expression. A greater understanding of how shear level influences specific remodeling events within each wall layer should aid in the development of targeted therapies to manipulate the remodeling process in health and disease.
flow-dependent arterial remodeling; shear stress; endothelial nitric oxide synthase; endothelium; smooth muscle; adventitia
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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STRUCTURAL REMODELING of the vascular wall and/or network occurs in response to changes in hemodynamic stimuli (pressure or flow) or tissue injury (13, 37). Whereas arterial wall remodeling takes place during physiological conditions, it is also involved in many clinical conditions (13), including aging, atherosclerosis, collateral artery development, hypertension, and tumor growth. Learning to manipulate this process will enhance our ability to treat many diseases. Arteriogenesis or arterial growth is one area of active investigation with a goal to develop new treatments for arterial insufficiency (17, 51). Recent studies (20, 48, 49) have demonstrated that the small arteries and arterioles that form collateral pathways are the sites of major hemodynamically significant adaptations subsequent to arterial occlusion, suggesting that therapy to promote arteriogenesis would be a more efficient treatment of arterial occlusion than angiogenic therapy (17). Whereas current studies are investigating potential therapies, many questions remain, especially related to the processes that occur naturally (17). For example, it is not known why wall areas are increased in some (24, 46), but not all (14), arteries subjected to chronic elevation of blood flow. Understanding what determines the rate and magnitude of structural wall changes as well as the specific nature of these alterations within the various wall layers should ultimately assist in treatment development to regulate the remodeling processes.
Our laboratory has been interested in the arteriogenesis that occurs during collateral artery development. We have observed rapid luminal expansion (46, 48) in small resistance arteries that form collateral pathways after abrupt arterial occlusion. The growth of these arteries occurs in response to alterations in blood flow or wall shear forces rather than ischemia (20, 48). In these vessels, luminal expansion, endothelial cell proliferation, and smooth muscle cell hypertrophy occur during the first week following model creation (46) when shear forces are at their highest. As the shear level is returned toward normal levels, between 1 and 4 wk, the rate of luminal expansion is decreased and smooth muscle cell hyperplasia is observed (46, 48). These observations led to the formulation of two hypotheses: 1) both the rate and magnitude of luminal expansion is dependent on the degree of initial shear elevation, and 2) specific remodeling events within the arterial wall layers depend on the level of shear alteration.
In this study, a modification of our original model was made to alter the perfusion territory of mesenteric arteries to create three levels of altered arterial blood flows and shear forces in identical arteries within the same animal. Luminal dimensions, wall areas, nuclear densities, and endothelial nitric oxide synthase (eNOS) were measured to investigate how the level of altered shear impacts the rate of remodeling, wall structure, and endothelial gene expression.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental design.
The Indiana University-Purdue University at Indianapolis Institutional
Animal Use and Care Committee approved all animals and procedures used
in this study. A vascular model (Fig. 1)
was created to produce multiple levels of blood flow and shear rate within the mesenteric arteries of adult male Wistar rats (~200 g). An
acute group (n = 8) was used to determine the initial
level of blood flow and wall shear rate immediately following model creation. Two chronic groups were used to evaluate the luminal diameter, wall remodeling, and eNOS expression at 2 (n = 7) and 7 (n = 10) days following model creation. All
animals were euthanized with an overdose of anesthetic and aortic
transection.
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Acute group.
After rats were anesthetized (100 mg/kg thiopental ip), 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 a laparotomy was performed, the ileal portion
of the bowel and the cecum were gently pulled into a support chamber
and bathed in 37°C phosphate-buffered saline (PBS). Arterial
ligations were then made to alter the number of first-order intestinal
arterioles perfused by the selected arteries to ~50%, 200%, and
400% of their preligation state. Separate experiments indicated that,
for those sections of bowel between marginal artery ligations,
essentially all the perfusion of the bowel was from the experimental
feed artery rather than through connecting pathways formed by small
intramural arterioles. Thus arterial blood flow was assumed to be
equivalent to the total tissue blood flow in the appropriate intestinal
sections. Tissue blood flow was measured by use of standard fluorescent
microsphere techniques described for rats as previously reported
(46). 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 microspheres/ml red fluorescent;
Molecular Probes) were injected through the carotid cannula, followed
by 0.3-ml flush of physiological Ringer solution. Approximately 0.5 ml
of blood was obtained during a 90-s collection period. Resting arterial
diameters were then measured with videomicroscopy techniques. Average
wall shear rate (WSR) was calculated according to the following
formula: WSR = (4Q)/(
r3), where Q is
ileal artery blood flow (ml/s) and r is arterial radius (cm).
Chronic studies.
Similar procedures for repeated observation of the mesenteric arteries
have been described elsewhere (46, 48). After model creation (Fig. 1), maximally dilated (10
4 M adenosine and
105 M sodium nitroprusside) diameter measurements of the
experimental and control (100%) arteries were recorded. At the final
time of observation (2 or 7 days), the rats were anesthetized and
maximally dilated diameters were recorded.
Histology and morphometry.
After the final diameter measurements, the aorta was cannulated with a
double-lumen cannula just superior to the bifurcation of the common
iliac arteries to perfusion fix the mesenteric arteries. The
double-lumen cannula allowed aortic pressure to be monitored during
perfusion. Before perfusion, the renal pedicles and subdiaphramatic aorta were ligated, and the left renal vein was partially transected proximal to the renal pedicle ligation to allow outflow of the perfusate. The ileal arteries were cleared with 30 ml of PBS
(~37°C) that contained 10.0 mg of the fluorescent DNA stain
Hoechst-33342 and a dilator cocktail (104 M adenosine and
105 M sodium nitroprusside). Once cleared, the arteries
were perfusion fixed with 10% neutral buffered formalin (~37°C)
for ~20 min at an intravascular perfusion pressure of 100-120
mmHg. The mesenteric arteries and bowel were then excised and placed in
fixative for ~24 h. A section from each artery (~0.5 cm) was
isolated from its mesenteric vascular bundle for fluorescent imaging
and plastic embedding. The remaining section (~0.5 cm) of each artery
was left in the bundle and embedded in paraffin.
Immunohistochemistry. Tissue sections were deparaffinized with xylene, rehydrated in graded ethanols, rinsed in purified water, and then washed in PBS (3× 5 min each). Heat-induced epitope retrieval was then performed in a pressure cooker with antigen retrieval solution (Vector) at 120°C, followed by a cooling period. After the PBS washes, endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide in PBS for 30 min at room temperature. Nonspecific binding was blocked by incubation in 1.5% normal horse serum (NHS) with 0.2% Triton X-100 in PBS for 20 min. The sections were then incubated with a mouse monoclonal Ki67 antibody (IgG1, 1:100; Novocastra Laboratories) or eNOS (IgG2a, 1:16,000) primary antibody (N30020-eNOS, Transduction Laboratories). Primary antibody was diluted in 1.5% NHS, applied to tissue section, and incubated overnight at room temperature in a humidified chamber. After the PBS washes, the sections were incubated with secondary biotinylated horse anti-mouse antibody (1:100; Vector) for 60 min at room temperature. Avidin binding and diaminobenzidine tetrachloride (DAB) chromogen staining were then completed using the Vectastain ABC-Elite Kit (Vector Laboratories). The sections were then dehydrated and mounted. Primary antibody was replaced with blocking serum to confirm that staining was specific to the antigen of interest. No immunostaining was observed on slides without primary antibody.
Densitometric analysis of NOS immunohistochemical labeling was performed similar to methods described previously (34). Digital images of arterial cross sections were acquired under green-wavelength illumination. The images were then imported into an image analysis program (Metamorph, Universal Imaging). A shading correction procedure (Specimen Gray Value
Background Gray
Value)/(White Reference Image Background Gray Value) was
performed on all images to compensate for any uneven field
illumination. Optical density (OD) measurements were then made
using gray-level thresholding techniques. OD was calibrated using a
stepped optical density filter (Edmund Scientific). Vessel images were
gray level thresholded to an OD level that corresponded to specific NOS
staining. Specific staining was considered to be the OD levels above
the maximal OD obtained from images of sections without primary
antibody. NOS content (arbitrary units) was determined by multiplying
the average OD and the area of the thresholded region within the
arterial wall.
Immunoblotting. In an additional group of animals, 100% and 400% arteries were isolated and immediately stored in liquid nitrogen. The frozen arteries were homogenized in RIPA buffer (10 mM NaH2PO4, pH 7.2, 150 mM NaCl, 2 mM EDTA, 50 mM NaFl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS). Equal amounts of protein (10 µg/lane) were loaded onto a 6% polyacrylamide gel and transferred to nitrocellulose at 80 mA, in 25 mM Tris, 192 mM glycine, 0.05% SDS, and 20% methanol overnight. Protein concentrations were determined using the bicinchoninic acid method (Pierce). Blots were blocked in 10 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 (TBST) containing 2% gelatin, 30 mg/ml casein, 5 mg/ml polyvinylpyrrolidine-40, and 0.5 mM EDTA for 2 to 4 h. Anti-eNOS antibody (1:2,000, Transduction Laboratories) was incubated with the blots overnight. After extensive washing in TBST, blots were incubated with horseradish peroxidase conjugated to anti-mouse IgG. Specific eNOS protein was detected using chemiluminescence according to the manufacturer's directions (Pierce Dura) and quantitated using a chemiluminescence imaging system (Fuji LAS-1000).
Data analysis.
All data were entered into a spreadsheet, the means ± SE
calculated, and figures or tables created. Mixed factorial ANOVA with
two repeated factors (vessel type and time of observation) was used for
comparison of in vivo diameters, followed by contrast comparisons to
evaluate significant main effects or interactions. All other variables
were analyzed with one-way or two-way (one-factor repetition)
repeated-measures ANOVA followed by the Bonferroni t-test to
make specific comparisons. The null hypothesis was rejected at
P 
0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hemodynamic data.
Averages of blood flow and wall shear rate for the different artery
types within acute animals are presented in Fig.
2. Compared with the 100% (control)
arteries, blood flow decreased 24 ± 9% in the 50% (reduced
flow) arteries and increased 159 ± 28% and 281 ± 52% in
the 200% and 400% (elevated flow) arteries, respectively. As
expected, a similar trend was observed for shear rate. Compared with
controls (100% vessels), shear rate decreased 39 ± 17% in the
50% arteries and increased 83 ± 28% and 200 ± 89% in the
200% and 400% arteries, respectively. Blood flow uniformly increased from the reduced flow artery to the highest flow artery in all animals.
A significant linear relationship was observed between the change in
perfusion territory and the percent change in blood flow (P 0.001; r2 = 0.74) and shear rate
(P 0.001; r2 = 0.45).
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Luminal dimensions and wall areas.
Maximally dilated arterial diameter measurements of each artery type in
the 2- and 7-day groups are reported in Table
1. For each vessel type, the average
percent change in diameter from the time of model creation to 2 or 7 days later and the average daily rate of change as percent from
original diameter are reported in Fig. 3,
A and B, respectively. No change occurred in the
diameter of the control (100%) arteries. The 50% arteries had a
reduced luminal diameter at 2 days (Table 1), which was not further
decreased at 1 wk (Fig. 3A). Consequently, the greatest rate
of change in diameter of the 50% arteries occurred during the first 2 days (Fig. 3B). The highest flow (400%) arteries also were
characterized by a significant diameter change at 2 days (Table 1). For
these 400% arteries, the rate of diameter change was similar during the first 2 and 7 days (Fig. 3B) resulting in a greater
percent increase at 7 days than at 2 days (Fig. 3A). The
200% arteries did not experience a significant increase in diameter
until day 7 (Fig. 3A, Table 1), and the rate of
diameter change in these vessels was greater after day 2 (Fig. 3B). For arteries subjected to elevated flow, both the
magnitude (Fig. 3A) and the rate (Fig. 3B) of the
diameter change were greater in the highest flow (400%) vessels.
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Cellular density.
Averages of nuclear counts (number per unit wall area) from each layer
(intima, media, and adventitia) within the arterial wall are presented
in Fig. 4, A-C.
Statistically significant changes were observed only in the 400%
arteries. Relative to the same animal controls (100% arteries), the
number of nuclei per unit wall area was increased 26 ± 9% and
32 ± 5% in the intima at days 2 and 7,
respectively, and 44 ± 10% in the adventitia at day
2; no differences were observed in the media. Positive Ki-67 (a
cell proliferation marker) staining within the intimal and adventitial layers of the 400% arteries of the 2-day group (Fig. 4D)
suggests that cellular proliferation accounts for much of the increase.
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NOS expression.
Micrographs and plots in Fig. 5,
A and B, demonstrate eNOS staining and present
the average endothelial eNOS content of the vessel types at 2 and 7 days following model creation. As can be seen from the images (Fig.
5A), eNOS staining was present only within the endothelium.
It is apparent from these micrographs that eNOS staining varies with
vessel type. The greater staining intensity of the 200% and 400%
vessels was a consistent observation within the animals. This
observation is supported by quantitation of the immunohistochemical
slides (Fig. 5B) and of immunoblots (Fig. 5C).
eNOS content normalized to luminal circumference was greater in the
400% than in the 100% arteries at both day 2 (289 ± 115%, P 0.001) and day 7 (559 ± 157%,
P = 0.041). This difference in eNOS content was
confirmed with Western blotting of 100% and 400% arteries at 2 days
(3.7 ± 1.2-fold increase, Fig. 5C). The increase in
eNOS content is much greater than the increase in endothelial cell
density (26-32%, Fig. 4), indicating that cellular eNOS content
is elevated. Shear-level dependent expression of eNOS is indicated by a
significant linear relationship between vessel type and intimal eNOS
content at 2 days determined by simple regression analysis (eNOS:
r2 = 0.69, P < 0.001).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The vascular ligation model utilized in this study was developed to address the question of how shear level influences arterial remodeling. It is a modification of the model previously developed in our laboratory (11, 46, 48) to study flow- or shear-mediated collateral artery development. A unique feature of this preparation is that several identical arteries are available to serve as control and experimental vessels. The primary stimulus is associated with the alteration of blood flow, presumably through changes in wall shear force, as the arterial ligations alter flow in the experimental vessels without significantly influencing arterial pressure (43, 48). In this modified model, we demonstrate that alteration of perfusion territory (Fig. 1) is positively correlated with blood flow and wall shear rate (Fig. 2). A distinct advantage of the model is that blood flow and shear force can be varied over a physiological to pathophysiological range from the normal in vivo state. In this, as well as previous studies (46, 48), paired diameter measurements of control (100%) arteries at the times of model creation and final experimentation are similar (Table 1, Fig. 3A). This constancy of control (100%) artery diameter, in addition to evidence that microvascular growth and capillary density are unchanged with careful handling of the bowel and mesentery (44, 45, 47), demonstrate that arterial remodeling within this model is due to hemodynamic alterations rather than the surgical procedures or manipulation of the preparation. Therefore, we believe this model offers a unique method for investigating shear level-dependent structural and biochemical remodeling events in a within subject design in intact animals.
Effect of shear level on luminal expansion. Previous studies have demonstrated that luminal expansion in large arteries continues until wall shear stress is normalized (21, 46, 54). It has been suggested that luminal expansion may occur only after wall shear exceeds a threshold level (6). We are not aware of previous studies that have directly addressed the rate of expansion at different shear levels. Earlier studies from our laboratory have suggested that the rate of diameter change is dependent on the flow or shear stimulus (43, 46, 48); however, this interpretation was based on an assumption of a gradual reduction in the stimulus along the collateral path (46, 48) and observations in different branching orders of arteries (43, 46, 48). It is also possible that luminal expansion proceeds at a constant rate within similar arteries after shear elevation until the expansion has restored shear to normal levels. Such a uniform rate of expansion is one potential explanation for the observation by Kamiya and Togawa (21) that wall shear was restored to normal levels after 6-8 mo in most carotid arteries but not those experiencing the greatest increases in blood flow. The current data obtained with identical (same branching order) resistance arteries clearly demonstrate that over moderate or physiological levels of altered flow or shear, the rate of luminal expansion is influenced by the level of the flow or shear stimulus (Fig. 3B).
Together with our earlier studies (48), the data now indicate that the greatest rate of luminal expansion occurs early and then diminishes as shear level is reduced by luminal expansion (46). An interesting observation was that the rate of diameter change was equal in magnitude for 50% and 400% arteries and that the diameter reduction in the 50% arteries was apparently complete by day 2 (Fig. 3, A and B). This is consistent with the suggestion by Brownlee and Langille (6) that arteries of mature animals are more sensitive (in terms of remodeling) to reduction than elevation of blood flow.Effect of shear level on wall remodeling. Unthank and colleagues (46) speculated that specific remodeling events depend on the level of the shear stimulus. However, some events associated with arterial remodeling are transient or sequential in nature (36, 42), and our previous observation that endothelial proliferation preceeds smooth muscle cell hyperplasia could simply represent the natural progression during shear-mediated remodeling. The current study provides compelling support for the premise that specific wall remodeling events, including cell proliferation, are dependent on the level of the shear stimulus. Flow-dependent luminal expansion (~17%, Fig. 3A) occurred in the 200% arteries without a significant increase in intimal or medial wall areas (Fig. 3, C and D) or in cell density within any wall layer (Fig. 4). In contrast, the luminal expansion within the 400% arteries (~33%, Fig. 3A) was characterized by increased intimal and medial areas (Fig. 3, C and D) and intimal and adventitial cell densities (Fig. 4). It is important to note that these wall changes in the 400% arteries were already apparent at 2 days (Figs. 3 and 4) when luminal diameter had only increased about 10% (Table 1, Fig. 3A), indicating that these aspects of wall remodeling are not simply dependent on the extent of luminal expansion. Whereas previous studies have demonstrated that changes in wall areas and cell densities are altered by an increase or decrease in blood flow, this is the first to demonstrate that these specific remodeling events associated with flow-mediated luminal expansion are dependent on the level of the flow or shear stimulus.
The observed increase in nuclear number per unit intimal and adventitial wall area in the 400% arteries clearly indicate that cells were added to these layers; otherwise, nuclear counts would be decreased in proportion to the degree of luminal expansion. Whereas cell migration could have contributed to the increase in cell number, positive Ki-67 staining within the intimal and adventitial layers of the 400% arteries of the 2-day group (Fig. 4D) suggests that cellular proliferation accounts for much of the increase. In addition, it is unlikely that migration contributed to the increase in cell number within the intima because the orientation, shape, and size of the nuclei within the intima was homogeneous and characteristic of endothelial cells (3, 4). The observation that the nuclear densities in the 50% and 200% arteries are similar to controls (100% arteries) may indicate that some degree of cellular apoptosis and proliferation occurs in these vessels; otherwise, luminal retraction would be expected to increase and luminal expansion decrease the nuclear density if changes in cell numbers were not occurring. Our observation of adventitial cell proliferation and increased cell density (Fig. 4, C and D) in the highest flow arteries confirm the important observation of Pourageaud and De Mey (32) that adventitial alterations occur during flow-mediated remodeling. Together with earlier studies (24, 46), this observation indicates that all vascular wall layers participate in the remodeling of resistance arteries in response to altered flow or shear. These observations raise fundamental questions that remain unanswered. A primary question concerns the mechanisms by which the elevation of blood flow results in the remodeling of the entire arterial wall. Over a decade ago, Langille et al. (22, 23) demonstrated that an intact endothelium is required for flow-mediated remodeling, and they proposed that endothelial cell products were involved in the chronic structural adaptations to altered flow. Whereas flow-dependent monocyte infiltration (2) and alteration of circumferential wall stress (43) might also be involved, multiple mechanisms exist through which endothelial cell products could mediate medial and adventitial remodeling events subsequent to changes in flow or shear. Given the multiple effects that nitric oxide can have (reviewed above), it is almost certain that the diffusion of this molecule from the endothelium to the media and adventitia of the vessel wall will influence both vascular cells and the extracellular matrix. The expression of other endothelial-derived substances, including growth factors such as basic fibroblast growth factor (36) and transforming growth factor-
1
(10), is altered by shear and might diffuse from the
endothelium to other wall layers. Such molecules could increase
extracellular proteolysis and release additional growth factors from
the matrix. Additional studies are needed to investigate the specific
mechanisms by which alterations in luminal flow influence remodeling
within the adventitial and medial layers.
Effect of shear level on endothelial gene expression. The simplest and most apparent explanation for the shear level-dependent remodeling is the shear level-mediated regulation of endothelial genes that influence the expression or activity of growth modulators. Cell culture and in vivo studies have demonstrated that shear force is capable of modulating mRNA and protein levels of endothelial-derived growth factors (25, 26, 30, 33, 36, 42, 53). We chose eNOS to evaluate the shear level-dependent alteration of endothelial gene expression in vivo for several reasons. Increases in shear stress in vivo are associated with elevation of eNOS mRNA, protein, and NOS activity (28) as well as nitric oxide production (18). Studies of endothelial cells in culture have demonstrated shear level-dependent expression of eNOS mRNA (56) and nitric oxide production (29). The nitric oxide system is believed to have a significant role in arterial remodeling (35, 40, 41), and elevation of nitric oxide corresponding to shear level could potentially explain the remodeling events we have previously observed (46). Our findings that eNOS expression is correlated with shear rate (Fig. 5) supports the hypothesis that specific remodeling events are regulated by shear level-dependent endothelial gene expression. Such alterations in endothelial gene expression would be expected to alter the balance between growth promoters and inhibitors within the vascular wall and thereby influence specific wall remodeling events.
The shear level-dependent expression of eNOS observed in this report supports data from many other studies that indicate a significant role for nitric oxide in vascular growth (27) and shear-mediated remodeling (35). However, a recent report by Ceiler and DeMey (7) suggests that inhibition of the nitric oxide system does not prevent shear-mediated structural remodeling of rat mesenteric arteries. It is not clear how these studies can be reconciled. One possibility suggested by Ceiler and DeMey (7) is that other compensating mechanisms might be activated during nitric oxide deficiency. It is also worth noting that inhibition of the nitric oxide system was initiated on the day of model creation by subcutaneous delivery from osmotic minipumps. Given our observations that significant remodeling and gene expression occur by days 2-3 in our model (Refs. 42, 43 and Figs. 3-5), much of the observed remodeling could have taken place before the inhibitor had reached an effective level within the treated animals. Regardless, numerous studies have demonstrated that nitric oxide is capable of regulating transcription factor activation (5, 8), stimulating the proliferation of endothelial cells in vivo (55), inhibiting smooth muscle cell (39) and fibroblast (38) proliferation, modulating the activity of matrix enzymes (9, 31, 40), as well as having significant interactions with other growth factors (12, 15, 19, 52). These studies, together with many others that have shown that shear alters eNOS expression, including in vivo expression in arteries (28) and in a shear-dependent manner in vitro (56), make it almost inconceivable that nitric oxide does not have a significant role in shear-mediated remodeling. In summary, the ability to create multiple levels of altered flow or shear in identical arteries within the same animal provides a powerful and unique method to investigate the effect of stimulus level on arterial remodeling. The current study demonstrates that the rate of luminal expansion, specific remodeling events within the wall layers, and the degree of endothelial gene expression are influenced by the level of blood flow or wall shear alteration. These observations may explain differences in the remodeling process reported for arteries exposed to chronic elevation of wall shear force. Further investigation is needed to extend our knowledge of arterial remodeling in general and specifically for shear-mediated collateral artery development or arteriogenesis (17). Such studies will provide a basis from which not only to determine how shear-dependent remodeling is altered by such conditions as hypertension (16) and diabetes (1), but also for the development of targeted therapies.| |
ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Jennifer L. Stashevsky and Sharon B. Bledsoe with the histological and immunohistochemical procedures performed in this study.
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FOOTNOTES |
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This work was supported by American Heart Association Grant-In-Aid IA9804591 (to J. L. Tuttle) and National Heart, Lung, and Blood Institute Grant HL-42898 (to J. L. Unthank).
Address for reprint requests and other correspondence: J. L. Unthank, Dept. of Surgery, Indiana Univ. School of Medicine, WD OPW 548, 1001 West Tenth St., Indianapolis, IN 46202-2879 (E-mail: junthank{at}iupui.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 May 2001; accepted in final form 18 May 2001.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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)
were placed on mesenteric and marginal arteries as indicated to alter
blood flow in the experimental arteries. The ligations altered the
number of first-order arterioles to ~50, 200, or 400% of their
preligation state as shown.
