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. Author manuscript; available in PMC: 2009 May 1.
Published in final edited form as: J Vasc Surg. 2008 Mar 21;47(5):1039–1047. doi: 10.1016/j.jvs.2007.12.043

Effects of External Wrapping and Increased Blood Flow on Atrophy of the Baboon Iliac Artery

Seung-Kee Min 1, Richard D Kenagy 2, Joseph P Jeanette 3, Alexander W Clowes 4
PMCID: PMC2409145  NIHMSID: NIHMS48038  PMID: 18358668

Abstract

Objective

Increased blood flow causes neointimal atrophy, while relief of wall tension with an external wrap causes arterial medial atrophy. To study the effects of blood flow and wall tension separately and together, we applied tight or loose wrap on high flow- or normal flow-iliac arteries in baboons.

Method

Baboon external iliac arteries were wrapped with loose (LW) and tight (TW) fitting expanded polytetrafluorethylene (ePTFE) leaving part unwrapped (UW). A downstream arterio-venous fistula was constructed on one side to increase blood flow approximately two fold. The arteries were perfusion-fixed with 10% formalin after 4 (n=5) and 28 days (n=5).

Results

At 4 days, compared to the UW artery, the LW and TW normal flow artery showed significant medial atrophy (23% and 30, respectively; P<.05) and in the TW a loss of cells (27%; P=.02), but no change in cell density. At 28 days medial cross-sectional area was decreased by the TW and LW under normal (45% and 28%, respectively; P<.05) and high (43% and 29%, respectively; P< .05) flow. High flow did not alter the effect of wrapping nor did it affect UW medial area. At 28 days the normal and high flow TW media showed an insignificant loss of cells, but had increased cell density (47% and 30%, respectively; P<.05) suggesting preferential loss of extracellular matrix. Decorin was expressed at the late time only in the TW normal and high flow media and was associated with tight packing of the collagen (detected by picrosirius red staining).

Conclusion

Loose and tight fitting ePTFE wraps, which induce an inflammatory foreign body response, caused medial atrophy with loss of cells and extracellular matrix; the tight wrap was more effective. High blood flow did not prevent or augment medial atrophy.

Atrophy of vascular tissue is both a physiological and pathological phenomenon. Physiologic examples include regression of the primary plexus of capillary-like vessels formed during embryonic development, the post-natal reduction of infrarenal aortic diameter1,2, and the loss of pericytes that is an inherent component of any angiogenic program3. Pathologic examples include the medial and fibrous cap thinning observed in advanced atherosclerotic plaques and the medial thinning seen in aneurysms. Investigation of mechanisms of vascular atrophy may lead to the discovery of new targets for treating stenotic vein grafts or restenotic stented arteries. This approach would enable treatment of only those patients that develop problems after vascular reconstruction rather than treating all patients as required when prevention of neointimal hyperplasia is the strategy.

Previous studies have demonstrated that increased blood flow (i.e. shear stress) causes neointimal regression in expanded polytetrafluoroethylene (eEPTFE) aorto-iliac grafts by decreasing cell proliferation and by increasing cell death and extracellular matrix (ECM) degradation4–6. In contrast to the graft, the adjacent iliac artery dilates in response to high flow and does not undergo atrophy.

The normal iliac artery differs from the neointima in the eEPTFE graft in at least two ways. First, the iliac wall carries tensile stress while the eEPTFE, not the neointima, presumably carries the tensile stress of the graft. Second, the ePTFE induces a foreign body inflammatory response (accumulation of macrophages and T cells7,8). Therefore, we have tested the hypothesis that the iliac artery will undergo atrophy in response to a reduction of wall stress and the addition of inflammatory stimuli by using a ePTFE wrap.

MATERIALS AND METHODS

Baboon Model

Segments of 60 µm internodal distance, un-reinforced, polytetrafluoroethylene (ePTFE; GORE-TEX® material; generously provided by W.L. Gore & Associates, Inc., Flagstaff, AZ) graft material were used to wrap the external iliac arteries of 10 male baboons as shown in figure 1. The tight wrapped segment was made by wrapping the collapsed artery and a 2.5 mm diameter rod together and removing the rod after sewing together the edges of the graft material; the loose wrapped segment was left unsewn. Thus, the artery could not dilate beyond its normal diameter (2.5 mm). The control segment of artery proximal to the graft material was dissected free of surrounding tissue, but was left unwrapped. A superficial femoral artery to vein fistula (10 mm length) was created on one side to increase blood flow. After 4 or 28 days (5 animals each) unwrapped (proximal to the graft), tight-wrapped and loose-wrapped segments were removed after perfusion-fixation of the vessels with 10% formalin. Animals were given 3 doses of bromo-deoxyuridine (30mg/kg/dose, subcutaneous injection) at 17, 9, and 1 hour before necropsy4.

Figure 1.

Figure 1

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Experimental model of wrapping. PTFE graft material was placed around the external iliac arteries and the tight segment was made by wrapping the collapsed artery and a 2.5 mm diameter rod together and removing the rod after sewing the edges of the graft material together.

Animal care and procedures were conducted at the Washington National Primate Research Center in accordance with state and federal laws and under protocols approved by the University of Washington Institutional Animal Care and Use Committee and the Regional Primate Research Center. Animal care and handling complied with the "Guide for the Care and Use of Laboratory Animals" issued by the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC, National Academies Press, 1996.

Morphometry

Formalin-fixed tissue segments were cut transversely at the midpoint and embedded in paraffin. Histologic sections were stained with hematoxylin / eosin. Because the intima contained mostly endothelial cells and occasional smooth muscle cells, atrophy was not evident grossly. Medial wall area was determined as EEL area minus luminal area from the average of measurements from two midpoint sections of each arterial segment. Average values of nuclear density were determined by counting medial nuclei of two different sections from the midsection of each arterial segment. Nuclei were counted within the grid of a reticle eyepiece at 400X in all four quadrants of a cross section and then total nuclear number was determined by multiplying nuclear density by medial area.

BrdU and TUNEL staining

Proliferating cells in the vessel wall were detected with an antibody to the thymidine analogue bromodeoxyuridine (BrdU; Boehringer-Mannheim, Basil, Switzerland). Apoptotic cell death was determined with terminal deoxy nucleotidyl transferase-mediated deoxyuridine-triphosphate nick end labeling of fragmented DNA (TUNEL) on cross-sections of the paraffin-embedded arteries as before4.

Immunohistochemistry

Cross sections of the paraffin-embedded arteries were stained with antibodies for smooth muscle alpha actin (for smooth muscle; 0.05 µg/ml 1A4, Zymed Laboratories, San Francisco, CA), CD68 (for monocyte/macrophages; 0.16 µg/ml KP1, Dako Corp., Carpinteria, CA), versican (0.5 µg/ml 2B1, Seikagaku America Corp., East Falmouth, MA), decorin (0.5 µg/ml 6B6, Seikagaku America Corp., East Falmouth, MA), and the versican cleavage neoepitope (10 ng/ml JSCDPE6). As a negative control, sections were incubated with the same concentration of appropriate non-immune IgG. Species specific secondary biotinylated antibodies were used with the avidin-biotin-peroxidase method (Vector Laboratories, Burlingame, CA). Hyaluronan was detected using biotinylated hyaluronan-binding protein6. Cross sections were also stained with Movat’s pentachrome (for collagen, glycosaminoglycans, elastin and nuclei), Verhoeff’s (for elastin), picro-sirius red (for collagen fibrils), and Alcian Blue (with or without 0.3 M MgCl2 for heparan, dermatan and chondroitin sulfates or for these glycosaminoglycans plus hyaluronan, respectively). Picro-sirius red staining under polarized light was quantified as follows. The medial area of each cross-section at 200X was analyzed using the histogram function of Adobe Photoshop using the RGB color system. Luminosity of the red channel averaged over 80% of the combined luminosity of the red, green and blue channels and this did not change among the treatment groups. Therefore, the ratio of average red channel luminosity of tight- or loose-wrap to unwrapped for each animal was calculated to quantitate changes in picro-sirius red staining.

Statistical analysis

Values reported are the mean ± SEM. Statistical differences were tested with the paired student T test or the Wilcoxon signed rank test using the Bonferonni correction for multiple comparisons.

RESULTS

Morphometric Analysis

Blood flow and shear stress were increased about 2 fold by the arterio-venous fistula at both 4 and 28 days (table 1). At 4 days under normal flow, both the loose and tight wrapped segments exhibited decreased cross sectional medial area compared to the unwrapped artery (by 23% and 30%, respectively; table 2 and figure 2A), while neither wrap had an effect under high blood flow. The tight wrap under normal, but not high, flow also significantly decreased medial cell number by ~25% (table 2 and figure 2B). By 28 days medial cross-sectional area was decreased by both wraps under normal and high blood flow, but the tight wrap had the greatest effect (table 2 and figure 2A). The tight wrap increased nuclear density at 28 days under normal and high blood flow, but the loose wrap had an insignificant effect under normal flow and a marginal effect under high flow (table 2 and figure 2C). Intimal area was very small and at no time was there any change after wrapping (total intimal nuclear number for normal flow unwrapped and tight-wrap at 28 days was 560 ± 73 and 567 ± 83, respectively; n=5). These results support the conclusion that there is a significant loss of extracellular matrix in the media of wrapped vessels by 28 days. Cross-sectional areas of unwrapped normal flow and high flow arteries were the same at 4 and 28 days (0.525 ± 0.085 mm2 vs 0.435 ± 0.070 mm2 for 4 days and 0.485 ± 0.085 mm2 vs 0.589 ± 0.096 mm2 for 28 days, respectively)

Table 1.

Terminal blood velocity and shear stress measurements in the external iliac artery.

Normal Flow 4 days High Flow 4 days Normal Flow 28 days High Flow 28 days
Velocity - (cm/sec) 74.2 ± 7.9 157.6 ± 22.8 90.2 ± 8.4 151.8 ± 10.0
Shear Stress – dyne/cm2 37.5 ± 3.5 81.1 ± 10.5 46.5 ± 5.5 78.3 ± 5.5
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Values are the mean ± SEM.

Table 2.

Morphologic Results of the PTFE Wrap after 4 and 28 days.

NF-unwrapped NF-loose wrap NF-tight wrap HF-unwrapped HF-loose wrap HF-tight wrap
4 days
IEL area (mm2) 2.47 ± 0.56 2.08 ± 0.38 1.75 ± 0.39 2.13 ± 0.56 2.02 ± 0.47 1.28 ± 0.24
EEL area (mm2) 3.06 ± 0.58 3.04 ± 0.35 2.54 ± 0.32 2.98 ± 0.48 3.01 ± 0.42 2.09 ± 0.35
Media (mm2) 1.13 ± 0.21 0.97 ± 0.21 0.79 ± 0.15* 0.85 ± 0.22 0.99 ± 0.32 0.81 ± 0.25
Medial Nuclear Number 703 ± 159 567 ± 137 507 ± 111* 393 ± 74 472 ± 101 408 ± 80
Medial Nuclear Density (cells/ mm2) 4560 ± 822 4270 ± 640 4728 ± 799 3549 ± 685 3950 ± 831 4015 ± 714
28 Days
IEL area (mm2) 2.11 ± 0.52 2.18 ± 0.46 1.61 ± 0.30 2.75 ± 0.69 2.00 ± 0.50 1.66 ± 0.23
EEL area (mm2) 3.33 ± 0.58 3.04 ± 0.40 2.27 ± 0.29 4.04 ± 0.66 2.91 ± 0.41 2.38 ± 0.26
Media (mm2) 1.22 ± 0.12 0.86 ± 0.07 0.66 ± 0.04* 1.29 ± 0.16 0.90 ± 0.12* 0.72 ± 0.10*
Medial Nuclear Number 445 ± 81 373 ± 26 330 ± 17 465 ± 50 425 ± 65 378 ± 44
Medial Nuclear Density (cells/ mm2) 2451 ± 218 2823 ± 78 3467 ± 245* 2520 ± 190 3235 ± 278† 3286 ± 248*
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Results are the mean ± SEM, n=5

*

P<.05 vs. unwrapped

†

P≤.06 vs. unwrapped. NF – normal blood flow, HF – high blood flow, IEL – internal elastic lamina, EEL – external elastic lamina.

Figure 2.

Figure 2

Figure 2

Figure 2

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The effect of wrapping and high blood flow on iliac artery medial cross-sectional area (A), nuclear number (B) and nuclear density (C). The left panel is for arteries 4 days after wrapping and the right panel is for arteries 28 days after wrapping. Open and closed bars represent the loose and tight wrap respectively. Results are presented as the % of the unwrapped control. All values are the mean ± SEM (n=5; * P≤.05 vs unwrapped artery; † P≤.06).

Cell Proliferation and Death

BrdU staining could not be detected in any any sample (data not presented). Low and variable levels of TUNEL staining were observed at 4 days; there were no significant differences between the unwrapped and wrapped arteries under normal or high blood flow (figure 3A). At 28 days there were no TUNEL positive cells in any samples.

Figure 3.

Figure 3

Figure 3

Figure 3

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A) Quantitation of TUNEL staining of unwrapped (open bars), loosely wrapped (hatched bars) and tightly wrapped (solid bars) iliac arteries at 4 days (n=5). There were no significant differences. B) Quantitation of macrophages (CD68) in unwrapped (open bars), loosely wrapped (hatched bars) and tightly wrapped (solid bars) iliac arteries at 4 days (n=5). There were no significant differences. C) Photomicrograph of CD68 immunostaining of tightly wrapped artery at 28 days. Arrows, in order from top to bottom, point to the internal elastic lamina, the external elastic lamina, and the PTFE/adventitia border. 200X

Identification of Cells and Extracellular Matrix Components

Cells

The number of macrophages in the medial layer of the vessel was small and highly variable, and the trend for increased macrophages in the media of the wrapped arteries at 4 days under normal or high flow was not significant (figure 3B). No macrophages were detected in the media at 28 days. In contrast and as previously observed in baboon PTFE grafts9, many of the cells in the PTFE graft and some in the adventitia were CD68 positive (figure 3C). Finally, the vast majority of cells in the media of all arteries stained positive for smooth muscle alpha actin (data not presented).

Extracellular Matrix

Picro-Sirius Red staining of collagen under polarized light revealed more intense red birefringence in normal flow, tightly wrapped arteries at 28days (figure 4), but not at 4 days (data not shown). Although this trend was also observed in the high blood flow arteries with tight wraps, it was significant only in the normal flow, tight wrap group (table 3). Loose wraps did not alter staining intensity. This increase in sirius red birefringence is consistent with more tightly packed collagen10 suggesting the preferential loss of non-collagenous matrix.

Figure 4.

Figure 4

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Picro-Sirius Red staining for collagen under bright field (left panels) or polarized light (right panels) of unwrapped (top panels) or tight wrapped (bottom panels) arteries at 28 days. Arrows indicate the medial-adventitial boundary. 200X

Table 3.

Picro-sirius red staining of PTFE-wrapped arteries at 28 days

Loose wrap Tight wrap
Normal flow 1.25 ± 0.25 3.13 ± 1.16*
High Flow 1.13 ± 1.18 1.63 ± 0.38
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Results are the ratio of average red channel luminosity expressed relative to the unwrapped control and are the mean ± SEM. n=5

*

P<.05 tight wrap vs. unwrapped.

Various extracellular matrix components were examined to determine if there was a loss of particular molecules during atrophy. The levels of chondroitin/heparan sulfate glycosaminoglycan (Alcian Blue alone or Movat’s pentachrome stain; figure 5A) and hyaluronan (HABR; data not presented) staining were similar in the unwrapped and tightly wrapped arteries under normal or high flow. Versican core protein was variably present in streaks and patches of the media and in the intima, but was not consistently altered by wrapping or increased blood flow (data not presented). In addition, elastin fibrils stained black with Verhoeff’s elastic stain were scattered throughout the media and were more dense at the adventitial boundary in this muscular artery. Elastin fibrils were not altered by the tight wrap at either 4 or 28 days under normal (figure 5B) or high flow (data not presented). In contrast, decorin staining was increased in the deep media of tightly wrapped arteries at 28 days in both normal flow (Fig 5C) and high flow (data not presented) arteries. Decorin was only variably observed at very low levels in the media of unwrapped and loosely-wrapped arteries under either normal or high blood flow at 28 days. At 4 days, it was not seen in the media of unwrapped or wrapped arteries with normal or high blood flow.

Figure 5.

Figure 5

Figure 5

Figure 5

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A) Alcian Blue staining for glycosaminoglycans including hyaluronan (low MgCl) in a 28 day normal flow unwrapped artery (left panel) and a tight wrapped-artery (right panel; the asterisk indicates the PTFE wrap). 100X B) Verhoff’s stain for elastin in a 28 day normal flow unwrapped artery (left panel) and a tight wrapped-artery (right panel). 200X C) Immunohistochemical staining for decorin in a 28 day normal flow unwrapped artery (left panel) and a tight wrapped-artery (right panel; the asterisk indicates medial decorin staining). Arrows indicate the medial-adventitial boundary. 200X

DISCUSSION

Wrapping of the normal iliac artery in baboons with ePTFE caused medial, but not intimal, regression within 4 days. This atrophy is the result of the loss of cells and extracellular matrix. These data confirm the findings of Courtman et al.11, who found that a rigid perivascular polyethylene cuff around the rabbit aorta caused medial, but not intimal atrophy. Why the media develops atrophy but the intima does not is unclear. Clinically relevant medial atrophy and degeneration are observed in atherosclerotic vessels and aneurysms12–14. Intimal atrophy has been observed in baboon PTFE grafts under high blood flow4 and spontaneously in stented arteries after extended times in rodents, pigs and humans15–18. In these cases these are newly formed intimas in the context of a relatively non-compliant vessel.

Because we were unable to detect cell death at 4 days, a time at which cell number is decreased, it is presumed that most cell death occurred at earlier times. Changes in cell proliferation with wrapping were not expected, because normal arteries have rates of SMC proliferation below 0.1%19. Thus, unlike the baboon PTFE graft neointimal model of atrophy, inhibition of proliferation could not contribute to atrophy. By 28 days there was a further regression with a preferential loss of extracellular matrix as indicated by the increase in nuclear density. This was clearly observed in the presence of the tight wrap, but the tendency was apparent with the loose wrap as well. Since previous studies demonstrated that versican is lost during neointimal regression in the baboon ePTFE graft6, versican immunostaining was assessed in the present experiments. However, versican is a minor component of the normal iliac artery media and was not changed by the tight wrap. Trace amounts of the ADAMTS-mediated DPE cleavage fragment of versican were detected, but did not increase during atrophy (data not presented). No loss was observed in elastin, hyaluronan or glycosaminoglycans (heparan and chondroitin sulfates), but collagen staining with picrosirius red revealed more tightly packed collagen fibrils at 28 days. The increased medial expression of decorin observed at 28 days is consistent with this observation as decorin is involved in the maturation of collagen and the increased packing and diameter of collagen fibrils20–22. Decorin also inhibits collagen degradation23 and, after balloon injury in rats, reduces neointimal ECM volume by compacting collagen fibrils24. All of these data suggest that the loss of ECM with the tight wrap involves a loss of non-collagenous, non-elastin components.

Our data suggest that inflammation as well as wall strain may regulate arterial atrophy. The PTFE graft matrix, unlike the media of the wrapped artery, contains large numbers of macrophages. These macrophages can secrete products (e.g. TNFα and FasL25,26) that may diffuse into the media. Since the loose PTFE wrap had ~ 60% of the effect of the tight wrap on medial area, but probably did not relieve tensile stress from the vessel, it might be concluded that the foreign body response of the loose wrap was important. However, a medial atrophy response was observed in the rabbit carotid artery wrapped by another carotid artery, thus indicating the foreign body response is not required27. It is possible that the upstream tight wrap may have increased shear stress and thereby generated signals that affected the loosely wrapped artery to cause medial atrophy.

Previous experiments in our lab showed that a tight ePTFE external wrap of a vein graft in the rabbit carotid artery causes a reduction of total cross-sectional area, SMC volume and matrix volume28. The observation that external wrapping of the vein grafts causes reduction of wall area supports the hypothesis that increased wall tensile stress may be an important stimulus for wall thickening29,30. However, a role for increased shear stress resulting from decreased luminal diameter is also possible. We and others have observed intimal atrophy or inhibition of intimal hyperplasia as a result of increased shear stress4,31–34. Of interest, Courtman et al11 found that, while a tight wrap over a balloon-injured artery initially inhibits intimal hyperplasia, by 3 weeks the wrap causes medial atrophy and no effect on intimal hyperplasia. These results stand in contrast to the effects of loose, non-porous wraps of normal rat35 and mouse36,37 arteries, which are postulated to cause intimal hyperplasia by increased inflammatory cytokine production. Finally, a loose, macroporous wrap is proposed to inhibit vein graft hyperplasia by promoting adventitial growth38,39.

It was expected that increased shear stress associated with high blood flow might work synergistically with wrapping to promote vessel wall atrophy. Apenburg et al found that shear stress increases Fas/FasL-dependent SMC apoptosis in vitro40. In addition, high blood flow increases NO production33,34, which has been shown to cause SMC apoptosis41. Finally, arterial matrix metalloproteinase 2 and 9 are increased by high blood flow42,43, and increased MMP activity has been shown to increase cell death44,45. However, there was no effect of blood flow at day 28 in the baboon ePTFE wrap model, and at day 4 high blood flow actually delayed arterial atrophy‥ The mechanism for this latter effect is not known. One possibility might involve TGFβ, since it is induced by increased shear stress42,46, causes intimal hyperplasia after injury47 and under some circumstances inhibits SMC apoptosis48.

The Importance of the Non-Human Primate Studies

These studies on atrophy in wrapped arteries may be of significant clinical value, especially because they were conducted in non-human primates. That deliberate wrapping of human vein grafts, like baboon arteries, might suppress wall thickening and luminal narrowing has been proposed and is actively being investigated by Angelini and colleagues49. More importantly, from our point of view, is the possibility that specific mechanisms of wall atrophy and luminal restoration common to humans and non-human primates might be revealed through this investigation. An important example supporting this possibility is the ongoing work on the role of PDGF/PDGF receptor family of molecules in intimal hyperplasia and wall thickening. PDGF and PDGF receptor activation promote intimal hyperplasia in many species including baboon50, but specific blocking antibodies to human PDGF receptors only cross-react with baboon receptors and do not inhibit inhibit PDGF receptors in other species. These antibodies not only suppress intimal thickening but also induce intimal atrophy in baboon vascular grafts51. The relevance of these observations is supported by recent publications reporting dramatic reversal of pulmonary arterial thickening and pulmonary hypertension by Gleevec in patients unresponsive to other medications52,53. This is an exciting but decidedly preliminary observation and cannot be rigorously attributed to an anti-PDGF receptor effect since Gleevec blocks several tyrosine kinases including both isoforms of the PDGF receptor.

Given the likely relevance of the baboon models of medial and neointimal atrophy (arterial wrap, high flow PTFE graft) for predicting outcome in humans, we are now conducting microarray studies to define a specific set of genes common to both models that would be expected to play a significant role in vascular atrophy and therefore might be targets for pharmacological intervention. The long term goal is to develop pharmacological strategies to expand the lumen of reconstucted vessels that develop stenosis, a goal that we think is achievable.

Acknowledgements

We thank the staffs of the Washington National Primate Research Center for their assistance with the animal surgery and care. We thank John Sandy for the antibody JSCDPE and Tom Wight and Pam Johnson for the HABR staining.

Supported by National Institutes of Health HL30946 and RR00166.

Footnotes

Clinical relevance: Research in arterial restenosis has focused on the biologic mechanisms and pharmacologic approaches to the prevention of intimal hyperplasia. An alternative therapeutic approach might be to induce atrophy of established intimal hyperplasia. We have previously reported that high blood flow induces neointimal regression in baboon ePTFE grafts. Here we provide another model of vascular atrophy induced by external wrapping. The similarity between baboons and humans with regard to their vascular system and individual genetic heterogeneity makes these experiments of great relevance. Up- or down-regulated genes common to both models might be key regulators of vascular atrophy, and therefore suitable therapeutic targets for pharmacological treatment of established lesions.

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Contributor Information

Seung-Kee Min, Department of Surgery, Seoul National University, Seoul, Korea.

Richard D Kenagy, Department of Surgery, University of Washington, Seattle, Wash.

Joseph P Jeanette, Department of Surgery, University of Washington, Seattle, Wash.

Alexander W Clowes, Department of Surgery, University of Washington, Seattle, Wash.

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