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timal formation at this site has been linked to extremes ofshear stress or to high temporal and spatial shear gradientsthat have been detected in flow modeling studies.12-17 Aputative role for shear stress has been strengthened byobservations that local shear stress influences neointimalformation in other vascular disorders18-20; however, therehave been few mechanistic studies linking shear stress tograft failure largely because of a paucity of animal models.

Numeric and hydraulic models have demonstratedthat shear stresses imposed on the bed of the graft arehighly sensitive to anastomotic angle, blood flow rates(Reynolds number), and the relative sizes of graft and hostvessels,21-23 whereas the local geometry of the immediateanastomosis site and bending of the host or graft vesselout of the plane of the anastomosis are of secondaryimportance.24 The effect of angle of anastomosis on localhemodynamics has been particularly well studied; there-fore we reasoned that a surgical model that allowed con-trol over anastomotic angle would facilitate assessment ofhemodynamic factors in neointimal formation. We devel-oped a model of distal end-to-side anastomosis with rab-bit carotid arteries in which anastomotic angle could bevaried from less than 10 degrees to 90 degrees. An advan-tage of the model was that the Reynolds number for flow,a key determinant of local flow patterns, was approxi-mately 80, which was close to the value (100) chosen forstudy in a previous numerical analysis of the effects ofbranch angle on hemodynamics and shear stress distribu-tion at anastomosis sites.25 We report that anastomoticangle profoundly affects tissue responses on the bed ofhost vessels. Very acute angles lead to intimal proliferation,

Failure of surgical revascularization of coronary, cere-bral, and peripheral arteries remains high in spite ofimproved surgical techniques and, when prosthetic vesselsare used, improved biomaterial. Ten-year mortality rates forcoronary artery bypass range from 15% to 30%, dependingon age, sex, previous left ventricular dysfunction, and grafttype1-4; moreover, patients frequently undergo second orthird operations because of failed grafts.5

Examination of both human tissues and experimentalanimal models has implicated intimal thickening at the dis-tal anastomosis site as a major contributor to late graft fail-ure.6-11 At this site, intimal thickening occurs mostcommonly along the suture line and on the bed of the hostartery, that is, the region of the host vessel that is oppositethe outflow from the graft.11 Intimal hyperplasia along thesuture line has been attributed to surgical injury and tocompliance mismatch of the graft and host vessels. The bedis not subjected to these traumas; instead, consistent neoin-

From the Department of Laboratory Medicine and Pathobiology,University of Toronto, and the Toronto General Research Institute,a

and the Department of Pathology, University Health Network.b

Competition of interest: nil.Supported by research grant T-4083 from the Heart and Stroke

Foundation of Ontario and by the Medical Research Council of Canada(Canadian Institutes for Health Research).

Reprint requests: B. Lowell Langille, PhD, Toronto General ResearchInstitute, 200 Elizabeth St, CCRW 1-836, Toronto, ON M5G 2C4,Canada (e-mail: langille@uhnres.utoronto.ca).

Copyright © 2001 by The Society for Vascular Surgery and The AmericanAssociation for Vascular Surgery.

0741-5214/2001/$35.00 + 0 24/1/115815doi:10.1067/mva.2001.115815

Effects of anastomotic angle on vascular tissueresponses at end-to-side arterial graftsZane S. Jackson, BSc,a Hiroyuki Ishibashi, MD,b Avrum I. Gotlieb, MD,a and B. Lowell Langille, PhD,aToronto, Ontario

Objective: Hemodynamics has been implicated in the late failure of arterial bypass grafts, which frequently occurs at thedistal anastomosis site. This study was designed to assess the relationship between local hemodynamics and pathologicresponses of the distal anastomosis by manipulation of the angle of anastomosis of the graft, a major determinant oflocal hemodynamics.Methods: End-to-side anastomoses of the right carotid to the left carotid arteries of rabbits were performed at anasto-motic angles of less than 10 degrees (acute), 45 degrees (intermediate), or 90 degrees (right angle), and then theupstream left carotid arteries were ligated to simulate pathologic occlusion. We examined tissue responses on the wallof the recipient vessel opposite the anastomosis site (the bed), where unusual hemodynamic forces are imposed.Results: Three months after surgery, intimal thickening was observed on the upstream portion of the acute, and morerarely, the intermediate anastomoses only. Medial thinning caused by loss of cells and matrix, and an aneurysm-like dila-tion, was observed in the right angle and some intermediate anastomoses, but not in the acute anastomoses. En faceconfocal microscopy at 3 weeks after surgery revealed severe disruption of the internal elastic lamina in all anastomoticmodels. Zymography and Western immunoblotting demonstrated gelatinolytic activity, caused by expression and acti-vation of MMP-2, that was lowest in the acute anastomoses, higher in the intermediate anastomoses, and highest inthe right-angle anastomoses.Conclusions: We infer that very different pathologic changes to the vessel wall are elicited when local hemodynamics ismanipulated by altering the anastomotic branch angle. (J Vasc Surg 2001;34:300-7.)

whereas right angle anastomoses produce atrophy of thebed comprised of both cell loss and matrix degradation,and which culminates in aneurysmal dilation of the hostvessel.

METHODS

This work was approved by the Animal CareCommittee of the Toronto Hospital and was conducted inaccordance with the Guide for the Care and Use ofLaboratory Animals.

Surgical manipulations. Adult, male, New Zealandwhite rabbits, weighing a mean of 3.3 kg ± 0.4 kg (SD)were anesthetized by intramuscular injection of 0.8mL/kg xylazine (2 mg/mL) and ketamine (90 mg/mL).Full surgical anesthesia was maintained with continuousintravenous infusion of the anesthetic mixture at a rate of0.035 mL/min. After cutaneous injections of lidocaine,both common carotid arteries were exposed by a midlinecervical incision. For anastomoses constructed at approxi-mately 90 degrees, the right common carotid artery wasexposed laterally to the right sternohyoid muscle, whereasthe right carotid artery was exposed medially to this mus-cle for other anastomoses. Vessels were stripped of adven-titia at the intended suture sites: the distal right commoncarotid artery and a proximal segment of the left commoncarotid artery. After intravenous infusion of 800 units ofheparin, the right common carotid artery was ligatedproximal to the bifurcation, clamped at an upstream site,and cut just proximal to the ligation. The left commoncarotid artery was clamped on either side of the intendedanastomosis site, and an arteriotomy was made. Botharteries were flushed of blood with heparinized saline solu-tion, and the right carotid artery was anastomosed, end-to-side, to the left carotid artery with 8-0 nonabsorbablepolypropylene sutures. All anastomoses were performedunder a surgical microscope.

After completion of the anastomosis, the left carotidartery was ligated approximately 1 cm upstream from theanastomosis site to simulate pathologic occlusion. Thearteries were rinsed lightly with lidocaine to limitvasospasm, and the incision was closed in layers. Longisil0.5 mL (150,000 IU/mL benzathine penicillin G,150,000 IU/mL procaine penicillin G, 0.9 mg methyl-paraben and 0.1 mg propylparaben) was given by intra-muscular injection just before the surgery, and Buprenex0.1 mL (0.3 mg/mL buprenorphine hydrochloride) wasadministered subcutaneously approximately 1 hour aftersurgery.

Hemodynamic assessments. Blood flow was mea-sured in unmanipulated animals (n = 5) and in experi-mental animals 7 days after surgery (n = 6, 2 of eachanastomosis model). The animals were anesthetized, andcarotid arteries were exposed as described above.Ultrasonic transit time flow transducers of 2 to 3 mm weremounted on the host vessel downstream from the anasto-mosis and coupled to a flowmeter (Transonics modelT206; Transonic Systems, Ithaca, NY). Flows wererecorded on a Gould RS3400 chart recorder (Gould

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Electronics, Cleveland, Ohio). Low-pass filters on theflowmeter were set to record mean flow; however, the fil-ters were switched frequently to record pulsatile flow (cut-off frequency was set to 100 Hz) for brief periods toensure that high-quality phasic signals were beingrecorded. Mean flows recorded 10 minutes after the startof hemodynamic assessments were used for statisticalanalysis.

Vascular casting. One week or 3 months aftersurgery (n = 4 per group), animals were killed with slowintravenous infusion of 1.0 mL of the euthanasia solution,T-61 (Hoechst, 200 mg/mL N-[2-m-methoxyphenyl-2ethylbutyl-(1)]-2 hydroxybutyramide, 50 mg/mL 4,4´-methylene-bis (cyclohexyltrimethyl-ammonium iodide)and 5 mg/mL tetracaine hydrochloric acid), after theintravenous infusion of heparin (1000 units). Thedescending thoracic aorta was immediately cannulated in aretrograde manner, and Batson’s number 17 corrosioncompound (Polysciences, Warrington, Pa) was infusedinto the vasculature at a pressure of 100 mm Hg. After thecasting material had set (approximately 30 minutes), thecarotid arteries were excised and immersed in 25% sodiumhydroxide until all tissue was digested from the cast.

Tissue analyses. One week (n = 3 per group) or 3months (n = 6 to 8 per group) after surgery, animals werekilled with slow intravenous infusion of the euthanasiasolution 1.0 mL, T-61, after the intravenous infusion ofheparin 1000 units. The descending thoracic aorta wasexposed by a midline thoracotomy and cannulated in a ret-rograde manner, then the carotid arteries were perfusionfixed with 3% paraformaldehyde in phosphate-bufferedsaline solution for 20 minutes at a perfusion pressure of100 mm Hg. Anastomoses were excised and embedded inparaffin, and midline longitudinal sections were stainedwith hematoxylin and eosin. Images captured by use of aPulnix TMC-7 video camera (Pulnix America, Sunnyvale,Calif) that was mounted on a light microscope were trans-ferred to a computer imaging analysis system (C. Imaging,model 640; Compix, Inc, Tualatin, Ore). Medial and inti-

Fig 1. Diagram of intermediate anastomosis (anastomotic angle =45 degrees) identifying heel, toe, and bed. Lines with arrow headsdepict simplified flow pattern. Asterisk indicates stagnation point.

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mal cell counts, as well as measurements of medial andintimal wall thicknesses, were obtained along the bed ofthe anastomotic models (Fig 1).

Confocal microscopy of the internal elastic lamina.En face examination of the internal elastic lamina (IEL)was performed by use of laser scanning confocalmicroscopy.26 Three weeks after surgeries, animals werekilled, and the carotid arteries were perfusion fixed as pre-viously described (n = 6 to 8 per group). The anastomoseswere excised, graft vessels were removed, host vessels wereopened longitudinally, and the beds were mounted ontoglass slides, endothelium side up, and examined with aBio-Rad laser scanning confocal microscope (modelMRC-1024; Bio-Rad, Hercules, Calif ) equipped with akrypton/argon laser. Because elastin autofluoresces, nostaining was required. Optical sections were capturedthrough the full thickness of the IEL and digitally pro-jected to provide a full-thickness view. Images were thenanalyzed by use of the computer imaging analysis system(C Imaging, model 640, Compix, Inc). Surface area ofelastin was measured as a percentage of total surface areafor consecutive fields along the midline of the bed.

Gelatin zymography. One week after surgery, ani-mals were killed by infusion of 1.0 mL of T-61 (n = 3 pergroup). Anastomoses were excised, stripped of adventitia,flash-frozen in liquid nitrogen, and ground to a fine pow-der. Protein was extracted in lysis buffer containing 1%sodium dodecyl sulfate (SDS), phenylmethanesulfonyl flu-oride 100 mmol/L and leupeptin 10 mg/mL in Trisbuffer (pH 7.0) 45 mmol/L. Protein concentration wasdetermined with a Bradford assay, then equal amounts ofprotein from each extract were electrophoresed on a 10%SDS-polyacrylamide gel containing 0.1% type I gelatin(Sigma Chemical Co, St Louis, Mo). After electrophore-sis, gels were washed with 2.5% Triton X-100 and incu-bated overnight in Tris 0.05 mol/L with CaCl2 2.5

mmol/L and 0.02% NaN3. Gels were stained withCoomassie blue, and gelatin degradation was observed aswhite lytic bands.

Western immunoblotting. Proteins extracted asdescribed above were electrophoresed on a 10% SDS-polyacrylamide gel and then transferred onto polyvinyli-dene diflouride membranes (Bio-Rad). The membraneswere probed with monoclonal anti-human matrix metallo-proteinase–2 (MMP-2) purified immunoglobulin G anti-body. Membranes were incubated with peroxidase-labeledantimouse secondary antibody (Amersham PharmaciaBiotech, Baie d’Urfe, Canada), treated with enhancedchemiluminescence detection reagent (Amersham LifeScience), and exposed to Kodak X-OMAR x-ray film(Eastman Kodak, Rochester, NY).

Statistical analysis. Data are presented as means ±SE, and sample sizes are provided for all data. Intimal andmedial wall thicknesses, intimal and medial cell numbers,and surface area of elastin for different anastomotic angleswere compared with unmanipulated controls by use ofone-way analysis of variance (ANOVA) followed byDunnett’s tests. Differences were considered significant atP less than .05.

RESULTS

Characterization of the model. Surgical manipula-tion successfully achieved three different angles of anasto-mosis (Fig 2) that averaged 8 ± 1 degree, 43 ± 6 degrees,and 91 ± 3 degrees, as determined from vascular casts per-formed 1 week after surgery. These are henceforthreferred to as acute, intermediate, and right-angle anasto-moses, respectively. Minimal or no stenoses were observedat the suture sites for all of the models (Fig 2, A-C). Bloodflow rates (Q) measured 1 week after surgery averaged 29± 1 mL/min, which were not significantly different fromflow rates measured in control animals (26 ± 3 mL/min).Rabbit carotid arteries average approximately 2 mm indiameter (D)27; therefore, assuming a kinematic viscosityfor blood (ν) of 0.038 stokes,28 the Reynolds number (Re= 4Q/πνD) for time-averaged flow was approximately 80.

Histologic assessment of the distal anastomosis.One week after surgery, intimal accumulation of cells wasevident on the beds of the acute and the intermediateanastomosis models (Fig 3, B and C), but never in unma-nipulated carotid arteries (Fig 3, A). The lesion was notcontinuous at this time; instead, numerous focal areasalong the bed contained subendothelial intimal cells. Theendothelium remained intact, and histologic study of themedia was similar to unmanipulated arteries. The suben-dothelial cells were smooth muscle cells, as demonstratedby intracellular actin distribution in en face fluorescencestaining with rhodamine phalloidin (data not shown). Thebeds of right-angle anastomoses showed no intimal accu-mulation at 1 week after surgery; instead, the wall on thebed was attenuated, and histologic study of the mediaindicated loss of both cells and matrix (Fig 3, D).

Three months after surgery, well-established, continu-ous neointimas were observed in the upstream portion of

Fig 2. Methylmethacrylate casts of anastomoses sites from acute(A), intermediate (B), and right- (C) angled model prepared 1week after surgery. D, Cast of right-angle model 3 months aftersurgery. Note aneurysmal dilation of host vessel.

A B

C D

the beds of acute anastomoses (Fig 3, E). The hyperplas-tic intimas decreased in thickness at more downstreamsites, and no intimal thickening was observed past themidpoint of the bed (Fig 3, F). At 3 months, intermediateanastomoses produced neointimas that were restricted tosites upstream of the bed (Fig 3, G and H, Fig 4, A andB). Right-angle anastomoses produced no intimal thick-ening of the bed (Fig 3, I and J, Fig 4, A and B).

Histologic study at 3 months revealed medial thinningalong the bed of intermediate and right-angle anasto-moses that involved loss of cells and matrix. In sites ofextreme medial thinning, the media was no longer orga-nized into lamellar units. Also, nonuniformity of nuclearprofiles suggested that smooth muscle cells were no longerconsistently circumferentially oriented (Fig 3, H). Sites ofthe most extreme medial thinning were devoid of medialcells (Fig 3, J). Both the magnitude and distribution ofmedial thinning varied according to anastomotic angle.The right-angle anastomoses displayed the most extrememedial thinning (Fig 3, I and J), and medial thickness wassignificantly reduced at all sites along the bed (Fig 4, Cand D). The intermediate anastomoses displayed normalmedial histologic conditions at upstream sites, except thatmedial thinning was observed at downstream sites (Fig 3,G and H, Fig 4, C and D). The acute anastomoses dis-played normal medial histologic conditions.

Vascular casts prepared at 3 months revealed thatluminal diameters of the host vessels were much enlargedat the graft site of right-angle anastomoses (Fig 2, D). Thisaneurysmal dilation was evident on gross dissection duringtissue harvesting in all right-angle anastomoses. Modestdilation was occasionally observed with the intermediatemodel but was never observed in the acute model.

Disruption of the IEL. En face confocal microscopicstudy of the IEL of the bed was performed 3 weeks aftersurgery. The IEL of control arteries formed a continuous,fenestrated layer of elastic tissue (Fig 5, A); however, therewas severe disruption of the IEL on the beds of anasto-moses performed at all anastomotic angles. Enlarged fe-nestrae and extensive tearing of the IEL was observed. Formost fields along the beds, only small islands of elastinpersisted (Fig 5, B and C), and some fields were devoid ofdetectable elastin. Surface area occupied by elastindeclined to approximately one half of that observed incontrol arteries (Fig 6). In the right-angle model, signifi-cant elastin disruption occurred at sites further upstreamthan in the intermediate and acute models.

Gelatin zymography and Western immunoblot-ting. Gelatin zymography of protein extracted from hostarteries 1 week after surgery revealed lytic bands at 70 and62 kDa in all anastomotic tissues (Fig 7), and Westernimmunoblotting identified these bands as the latent andactive form of MMP-2, respectively, (data not shown).Although the effects of anastomotic angle on MMPexpression and activity were modest, the right-anglemodel produced the largest amount of active MMP-2, theintermediate model generated less active enzyme, and theacute model produced the weakest activation of MMP-2.

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A faint lytic band at 89 kDa, probably because of MMP-9,was most prominent in tissue from the right-angle model.

DISCUSSION

The most striking findings of this study were that vas-cular grafts constructed with low anastomotic anglesdeveloped neointimal thickening on the bed of the hostvessel, whereas grafts with anastomotic angles close to 90degrees displayed thinning of the vessel wall and aneurys-mal dilation of the host artery at this site. These findingssuggest that pathologic outcome of grafting procedures inthe clinical setting may be highly sensitive to anastomoticangle. Intimal thickening at the bed region of graft out-flow regions has been reported previously,29 but we areaware of no demonstrations of wall atrophy/dilation withright-angle anastomoses. The latter may be due to thepaucity of studies examining large anastomotic angles invivo. In one previous study, Norberto et al30 observed ahigh rate of patency when polytetrafluoroethylene graftswere coupled to host arteries by interposition vein graftsthat met the host vessel at 90 degrees, but no aneurysmaldilation was reported. The segment of vein graft that wasperpendicular to the host artery was only 4 mm long,whereas the upstream segment of polytetrafluoroethylenegraft approached the native artery at an acute angle; con-sequently, local flow conditions are probably quite differ-ent from those prevailing in our study. Alternatively,absence of dilation in the previous study may reflect thedifferent materials used for the graft. Unfortunately, anattempt by Norberto et al30 to vary graft angle in onegroup of animals was not informative with respect to ourstudy because 80% of the grafts thrombosed.

Fig 3. Longitudinal, hematoxylin and eosin–stained sectionsfrom unmanipulated left carotid artery (A) and from bed of acute(B), intermediate (C), and right-angle (D) anastomoses prepared1 week after surgery (n = 3/group). E-J, Histologic sections,prepared three months after surgery, of upstream (E, G and I)and downstream (F, H and J) portions of bed from acute (E andF), intermediate (G and H) and right-angle (I and J) anasto-moses. Figures are representative of n = 6 to 8 animals per group.Arrows identify IEL.

A B C D E

F G H I J

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In large distributing arteries, extracellular matrix bearsmuch of the wall tension; therefore remodeling of matrix isa prerequisite for structural arterial dilation. We found clearevidence for matrix degradation at anastomosis sites thatwas manifest as gross disruption of the IEL and upregula-tion and activation of the gelatinolytic enzyme, MMP-2.Interestingly, however, dependence of these phenomenaon anastomotic angle was modest. IEL disruption at thebed was profound, but it was similar for all anastomoticangles. It is possible that deeper matrix was more affectedwith larger anastomotic angles. Disruption of the IEL inanastomoses constructed at low angles may facilitatemigration of medial smooth muscle cells to the intima.

MMP-2 upregulation was also marked for all angles ofanastomosis. Increased presence of the active form of the

enzyme was seen for the right-angle anastomoses, whichmay have contributed to greater matrix degradation at thissite, but the effect was not striking. It is likely thereforethat other matrix-degrading enzymes participate inremodeling of the media of the bed.

The hemodynamic conditions that predispose to inti-mal thickening versus atrophy and dilation were not deter-mined in this study, but previous flow modeling studies

Fig 4. Distributions of intimal thickness (A), intimal cell count (B), medial thickness (C), and medial cell count (D) of beds of acute(n = 6), intermediate (n = 8), and right-angle anastomoses (n = 6) at 3 months after surgery. Position refers to distance along bed fromposition directly opposite heel of anastomoses. Asterisk indicates (P < .05) difference between anastomoses and unmanipulated vessels(n = 4). ANOVA followed by Dunnett’s test.

A B

C D

Fig 5. Confocal photomicrographs of IEL of an unmanipulatedcarotid artery (A) and beds of right-angle (B), and acute (C)anastomoses prepared 3 weeks after surgery. Note extensive dis-ruption of elastin at anastomoses.

Fig 6. Spatial distribution of total elastin surface area within IELof beds of acute (n = 4), intermediate (n = 6), and right-angle (n= 4) models compared with unmanipulated arteries (n = 4). Dataare percent of elastin surface area present within microscope fieldrepresented as mean ± SE. Axial position is as defined in legendto Fig 4. Open symbols represent significant differences (P < .05)between experimental and unmanipulated vessels. ANOVA fol-lowed by Dunnett’s test.

A B C

provide important insights.21-25,31 Much work has beendone with steady flow conditions to model time-averagedflow phenomena; however, pulsatile flow introducesimportant effects that also must be considered.

Anastomotic flow patterns impose a stagnation pointon the bed of the host vessel, where flow divides into a for-ward flow (positive shear stress, directed downstream) dis-tal to the stagnation point and a proximal reverse flow(negative shear, directed upstream) that establishes a com-plex vortex flow pattern in the occluded stump of the hostartery (Fig 1). Shear stresses are zero at the stagnationpoint, and they are much lower at more proximal sitesthan are shear stresses at most other sites of the bed or inungrafted arteries. Shear stresses downstream from thestagnation point become higher than those observed inungrafted arteries, sometimes by severalfold. With steadyflow, increased angle of anastomosis shifts the stagnationpoint upstream; it is almost opposite the midpoint of thegraft artery for right-angle anastomoses, when flow ratesare typical of central arteries.25 At low anastomotic angles,the stagnation point moves downstream and can lie distalto the bed. The location of the stagnation point alsodepends on flow rate, especially with low anastomoticangle; consequently, the stagnation point migrates up anddown the bed region during the cardiac cycle.

The numerical analyses of Fei et al25 are particularlygermane to this work because they examined flow patternsat anastomoses of varying angles (20-70 degrees) for aReynolds number (Re = 100) that was close to the meanReynolds number of our in vivo model (Re ≅ 80). For lowanastomotic angles, they found that low mean shearstresses are delivered to the bed region opposite the graftorifice,25 where we observed intimal thickening. Thisobservation is consistent with previous findings that inti-mal thickening correlates with low shear in many settingsincluding atherosclerosis,32 intimal proliferation afterexperimental injury,33 and when flow rates are manipu-lated in vascular grafts.34,35 The sites of intimal thickeningare also subject to large temporal variations in shear stress.Fei et al25 examined only steady flow conditions; however,Ojha36 studied pulsatile flow through hydraulic models of30-degree anastomoses, at a slightly higher Reynoldsnumber (mean Re = 185, peak Re = 925). He showed thatthe stagnation point migrates from upstream of the heelalmost to the toe of the graft during the acceleration phaseof the flow cycle. These effects are accentuated at loweranastomotic angles, so we anticipate that large fluctuationsin shear stress are imposed on sites of intimal thickening inour low-angle grafts. This observation is also consistentwith previous findings that intimal thickening also local-izes to sites of fluctuations in shear stress.37

The hemodynamically induced atrophy of the arterywall that we observed in right-angle anastomoses has notbeen reported previously at anastomosis sites. Medialthinning, cell loss, and aneurysmal dilation of the arterywere localized to the vicinity of the graft site and imme-diately downstream from this location. Fei et al25

reported that high anastomotic angles produced maxi-

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mum spatial gradients in shear stress and maximum shearstresses in the bed region. The latter were nearly triplethose observed in ungrafted arteries. In contrast, 20-degree anastomoses produced peak shear stresses thatwere less than 50% more than normal shear stress.Pulsatile flow imposes large temporal gradients in shearstress that are accentuated by the movement of the stag-nation point and the site of peak shear stress along thebed during the cardiac cycle; however, the displacementof the stagnation point is most modest for large anasto-motic angles. It appears therefore that high shear stressand high spatial gradients of shear correlate with the wallatrophy that characterized the bed of right-angle anasto-moses. These features of the shear stress distribution onthe bed are probably sensitive to other features of graftgeometry, and it may be possible to further assess theirimportance by alternative experimental manipulations.For example, increasing the area of the graft orifice wouldbe expected to distribute shear stresses more widely andreduce peak shear gradients. The effects of such modifi-cations could be assessed in future studies.

Hemodynamically produced aneurysmal dilation ofarteries also occurs downstream from stenoses (post-stenotic dilation [PSD]). Originally, it was believed thatturbulence causes PSD formation38; however, we demon-strated that PSDs could be produced in rabbit carotidarteries at Reynolds numbers that were well below turbu-lent values.39 Our previous flow modeling studies demon-strated that aneurysmal dilation occurred at sites where astagnation point (referred to as the reattachment point forpoststenotic flows) sweeps up and down the vessel with thecardiac cycle. High peak shear stresses and high spatialgradients of shear39 mimic those that occur on the bed ofgrafts constructed at large angles. Given the very different

Fig 7. Gelatin zymography of protein extracts from controlcarotid artery and bed of acute, intermediate, and right-angleanastomoses prepared 1 week after surgery (n = 3/group).

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geometries of stenoses and end-to-side anastomoses, it islikely that one or more of these phenomena are responsi-ble for the tissue responses that lead to dilation.

The intercellular signaling that elicits the remodelingwe have observed was not studied. However, endothelialcells drive most vascular tissue responses to altered shearstress, and recent studies have produced significant insightsinto the biologic responses of endothelium to the highlocal shear stress and high shear gradients that characterizestagnation/reattachment sites. Cultured endothelial cellsnear flow stagnation points, when compared with cellsexposed to no or uniform shear stress, exhibit increased cellproliferation, cell migration, and increased activation ofmultiple signal transduction pathways, as indicated by highlevels of nuclear NF-κB, Egr-1, c-Jun, and c-Fos.40 It is notclear which targets of these or other pathways drive the tis-sue responses that we see at right-angle anastomoses, butsignaling to underlying smooth muscle cells is almost cer-tainly involved. One candidate mediator of interest is tissueplasminogen factor (tPA). The tPA is released by endothe-lium in a shear-sensitive manner, and a resulting produc-tion of plasmin may contribute to matrix degradationdirectly or indirectly via activation of MMPs.41-43 It wouldtherefore be of interest to examine tPA expression in thevicinity of stagnation points.

In summary, we have shown that tissue remodeling atend-to-side arterial anastomoses is highly sensitive to graftangle and presumably to the effects of anastomotic geom-etry on local hemodynamics. It is likely that these effectswill have an impact on long-term pathologic outcomes ofclinical grafting procedures.

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Submitted Sep 18, 2000; accepted Jan 12, 2001.

THE PACIFIC VASCULAR RESEARCH FOUNDATION

IS ACCEPTING APPLICATIONS

FOR THE

2002 WYLIE SCHOLAR AWARD IN ACADEMIC VASCULAR SURGERYThe Wylie Scholar Award was established by the Pacific Vascular Research Foundation to honor the legacy of

Edwin J. Wylie, by providing research support to outstanding vascular surgeon-scientists.The Award is intended to enhance the career development of academic vascular surgeons with an established

research program in vascular disease. The award consists of a grant in the amount of $50,000 per year for 3 years.Funding for the second and third years is subject to review of acceptable progress reports. This

3-year award is nonrenewable and may be used for research support, essential expenses, or other academic pur-poses at the discretion of the Scholar and the medical institution. The award may not be used for any indirect costs.

The candidate must be a vascular surgeon who has completed an accredited residency in general vascularsurgery and who holds a full-time appointment at a medical school accredited by the Liaison Committee onMedical Educators in the United States or the Committee for the Accreditation of Canadian Medical Schools inCanada.

The applications are due by February 1, 2002, for the award to be granted July 1, 2002. Applications may beobtained by writing to: Pacific Vascular Research Foundation, Wylie Scholar Award, 3627 Sacramento Street, SanFrancisco, CA 94118.