altered hemodynamics, endothelial function, and protein ... · altered hemodynamics, endothelial...

Altered hemodynamics, endothelial function, and protein expression occurwith aortic coarctation and persist after repair

Arjun Menon,1 Thomas J. Eddinger,2 Hongfeng Wang,1 David C. Wendell,3 Jeffrey M. Toth,1,4

and John F. LaDisa, Jr.1,5

1Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin; 2Department of Biological Sciences,Marquette University, Milwaukee, Wisconsin; 3Duke Center for Cardiovascular Magnetic Resonance, Duke University,Durham, North Carolina; 4Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin;and 5Department of Pediatrics, Children’s Hospital and the Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 29 May 2012; accepted in final form 20 September 2012

Menon A, Eddinger TJ, Wang H, Wendell DC, Toth JM, LaDisaJF Jr. Altered hemodynamics, endothelial function, and protein expres-sion occur with aortic coarctation and persist after repair. Am J PhysiolHeart Circ Physiol 303: H1304–H1318, 2012. First published September28, 2012; doi:10.1152/ajpheart.00420.2012.—Coarctation of the aorta(CoA) is associated with substantial morbidity despite treatment.Mechanically induced structural and functional vascular changes areimplicated; however, their relationship with smooth muscle (SM)phenotypic expression is not fully understood. Using a clinicallyrepresentative rabbit model of CoA and correction, we quantifiedmechanical alterations from a 20-mmHg blood pressure (BP) gradientin the thoracic aorta and related the expression of key SM contractileand focal adhesion proteins with remodeling, relaxation, and stiffness.Systolic and mean BP were elevated for CoA rabbits compared withcontrols leading to remodeling, stiffening, an altered force response,and endothelial dysfunction both proximally and distally. The proxi-mal changes persisted for corrected rabbits despite �12 wk of normalBP (�4 human years). Computational fluid dynamic simulationsrevealed reduced wall shear stress (WSS) proximally in CoA com-pared with control and corrected rabbits. Distally, WSS was markedlyincreased in CoA rabbits due to a stenotic velocity jet, which haspersistent effects as WSS was significantly reduced in correctedrabbits. Immunohistochemistry revealed significantly increased non-muscle myosin and reduced SM myosin heavy chain expression in theproximal arteries of CoA and corrected rabbits but no differences inSM �-actin, talin, or fibronectin. These findings indicate that CoA cancause alterations in the SM phenotype contributing to structural andfunctional changes in the proximal arteries that accompany the me-chanical stimuli of elevated BP and altered WSS. Importantly, thesechanges are not reversed upon BP correction and may serve asmarkers of disease severity, which explains the persistent morbidityobserved in CoA patients.

vascular remodeling; contractile protein; hemodynamics; endothelialdysfunction; nonmuscle myosin

COARCTATION OF THE AORTA (CoA) is one of the most commoncongenital cardiovascular anomalies and is associated withreduced life expectancy due to sources of morbidity, includinghypertension and early-onset coronary disease, despite im-proved postoperative outcomes (9, 26, 30, 44). Untreated CoAprovides mechanical stimuli for vascular remodeling in theform of elevated blood pressure (BP) proximal to the coarcta-tion and potentially adverse distributions of wall shear stress(WSS) throughout the thoracic aorta. Normotensive repaired

CoA patients have also demonstrated increased carotid intimal-medial thickness and compromised vascular function that ap-pears to persist upon clinical evaluation 12–20 yr after inter-vention (11, 72). The mechanism(s) for this compromisedvascular function, possibly involving endothelial function orthe expression of smooth muscle (SM) proteins affecting struc-tural and functional properties of the aorta, is not presentlyknown in these postrepair patients.

Vascular remodeling describes the response to physiologicalor pathological stimuli wherein blood vessels undergo com-pensatory changes in lumen diameter and wall thickness tomaintain wall tensile stress or WSS (8, 63). The vascularendothelium is dynamic, serving to sense WSS and regulatevasomotor tone via the release of dilatory substances as well asanti-inflammatory agents to maintain vascular health. Impairedvasorelaxation through endothelial nitric oxide (NO) release,known as “endothelial dysfunction,” is a characteristic effect ofhypertension in human and animal models including prior CoAmodels (16, 38). Along with vasorelaxation, arterial constric-tion mediated by arterial SM may also play a role in theadaptive response of arteries to hypertension, as alterations inthe resting and active contractile force of elastic conduitarteries, such as the thoracic aorta and carotid arteries, mayindicate atherogenic changes in stiffness (23). Mounting evi-dence also suggests these vascular impairments caused by theinterplay between altered vasorelaxation and vasoconstrictionare a major risk factor in the development of coronary arterydisease (40, 48).

In addition to the role of the endothelium alluded to above,the maintenance of arterial structure and the functions de-scribed above is critically dependent on the controlled expres-sion of contractile and structural proteins by fully differentiatedSM (51, 52). A change from the fully differentiated state(dedifferentiation) of SM has been demonstrated to occur inconditions of vascular injury, and these changes may thus bepresent in CoA (29, 31). These phenotypic changes may beevident through the expression of SM contractile marker pro-teins in proximal and distal locations within the descendingthoracic aorta (dAo), including SM �-actin, SM and nonmuscle(NM) myosin, and caldesmon. SM �-actin and SM myosinheavy chain are isoforms of contractile proteins and are excel-lent markers of the differentiation state of SM, whereas calde-smon is an actin- and calmodulin-binding protein in SM thatmay similarly serve as a marker of the differentiation state(52). NM myosin is a motor protein that plays a role in cellularforce and translocation through its actin-binding and contrac-tile properties. Although NM myosin is distinguished as an

Address for reprint requests and other correspondence: J. F. LaDisa, Jr.,Dept. of Biomedical Engineering; Marquette Univ., and Dept. of Pediatrics;Children’s Hospital of Wisconsin, 1515 W. Wisconsin Ave., Rm. 206, Mil-waukee, WI 53233 (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 303: H1304–H1318, 2012.First published September 28, 2012; doi:10.1152/ajpheart.00420.2012.

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NM protein, it is generally present in SM cells and has severalfunctions. Through development, differentiation is thought tooccur such that NM myosin expression decreases from �50%in embryonic stages to �10% in mature animals (19). How-ever, recent research has indicated that NM myosin canuniquely provide a convergence point between external forcesand cellular responses that regulate its activation (68), thusproviding a possible link between altered mechanical stimuliand the structural and functional changes associated withsources of morbidity commonly observed in CoA. Externalforces may result in phosphorylation or conformationalchanges in NM myosin heavy chain that subsequently altercytoskeleton mobility and filament interaction, ultimately lead-ing to changes in SM migration and adhesion (15). Whileseveral reports have documented a mechanosensitive role forNM myosin in cell culture experiments (33, 65), no studies todate have observed the expression of vascular NM myosin inpathophysiological conditions where indexes of hemodynam-ics and vascular biomechanics can be precisely quantified.Hemodynamics refers to the forces that govern blood flowthrough the cardiovascular system and are best represented bythe indexes of BP and WSS for the present investigation,whereas biomechanics is concerned with the effects producedby these forces exerted on the vasculature that can be quanti-fied through the indexes of cyclic strain, structure, and func-tion.

In addition to contractile proteins, cytoskeletal and extracel-lular contractile proteins associated with integrins, known asfocal adhesions, have been studied to understand their influ-ence on the regulation and contraction of SM (53, 57, 70).Talin is a cytoskeletal focal adhesion protein on the cytosolicside of the membrane, and fibronectin is an extracellularglycoprotein (20, 21). When considered together, expression ofthese contractile and focal adhesion proteins provides an ad-ditional indicator of SM differentiation beyond previous stud-ies, which observed only SM �-actin or SM myosin.

Previous research has characterized changes in pulse BP,aortic capacitance, and WSS occurring due to CoA (24, 35,36), but the specific mechanisms linking these changes to thelong-term morbidity observed in untreated and postrepair pa-tients have not been conclusively identified to date. The ob-jective of this work was to couple subject-specific computa-tional techniques with an established approach in rabbits thatmimics untreated CoA as well as vascular morphology typi-cally observed after resection with end-to-end anastomosis toquantify 1) the severity of hemodynamic and vascular biome-chanics alterations before and after correction and 2) endothe-lial function and the phenotypic modulation of vascular SMcontractile and focal adhesion proteins along with their rela-tionship to altered vascular structure, function, and stiffness.The data presented here suggest that hemodynamic and bio-mechanical alterations caused by CoA result in endothelialdysfunction, dedifferentiation of arterial SM, and medial thick-ening. The mechanical consequences include altered contrac-tile force and resting stiffness and a shift of the SM phenotypefrom contractile toward “synthetic,” with impaired vasorelax-ation via the endothelial NO pathway. Together, these func-tional and structural alterations may explain the high rates ofresidual morbidities seen in CoA patients despite BP restora-tion due to surgical correction.

METHODS

Experimental protocol. After approval from the Institutional Ani-mal Care and Use Committee, male New Zealand White rabbits (�10wk old and weighing 0.8–1.2 kg) were randomly designated toundergo proximal dAo CoA, resulting in a �20-mmHg BP gradientusing silk (permanent) or Vicryl (degradable) suture as previouslydescribed (43) to mimic untreated CoA and surgically corrected CoApatient populations, respectively. Importantly, the putative guidelinesuggestive of treatment for CoA in humans is a BP gradient of �20mmHg at rest (55). A control group (nonexperimental) was alsoestablished (n � 7 rabbits/group). Rabbits underwent extensive intra-operative monitoring for O2 saturation, heart and respiratory rate,temperature, and capillary refill time throughout all experimentalprocedures, which continued for 3–5 h after all surgical and imagingprocedures were completed (43).

MRI. Rabbits underwent imaging as previously described (43) witha 3-T Sigma Excite scanner (GE Healthcare, Waukesha, WI) using aquadrature knee coil to obtain magnetic resonance angiography(MRA) data for vascular geometry and phase-contrast MRI (PC-MRI)data for the determination of cyclic vascular strain and ascendingaortic blood flow for use with computational fluid dynamic (CFD)simulations. Mean and maximum Green-LaGrange strain (E�� andE��max, respectively) were calculated as follows: E�� 0.5[(Dcurrent/Ddiastolic)2 � 1] and E��max � 0.5[(Dsystolic/Ddiastolic)2 � 1], where Dis diameter (35). The aortic distensibility and pressure-strain elasticmodulus were also calculated using PC-MRI data (61). All imagingdata was obtained at the conclusion of the experiment, 1 day beforethe completion of the experiment at 32 wk of age. For corrected andCoA groups, this was �22 wk after the coarctation was induced.

BP and tissue harvest. Before tissue harvest at 32 wk of age, rabbitswere anesthetized for the simultaneous measurement and recording ofBP waveforms at the common carotid and femoral arteries via fluid-filled catheters (43). Arteries were then removed from the followingfour locations after euthanasia by an intravenous overdose of pento-barbital sodium (100 mg/kg): 1) the left common carotid, 2) the dAoabove the coarctation (proximal), 3) the dAo downstream of thecoarctation (distal), and 4) the femoral artery. Arteries were excisedwith extreme care to avoid damaging the endothelium or SM tissueand placed in either 10% neutral buffered formalin for histology orcold (4°C) physiological salt solution [PSS; containing (in mM) 140NaCl, 4.7 KCl, 1.2 MgSO4, 1.6 CaCl2, 1.2 NaHPO4, 2.0 MOPS(adjusted to pH 7.4), 0.02 Na2 EDTA (to chelate heavy metals), and5.6 D-glucose] in preparation for myograph and immunohistochemicalanalysis.

CFD simulations. Computational representations of the aorta andarteries of the head and neck were reconstructed from imaging data aspreviously described (35). Ascending aortic PC-MRI waveforms werethen mapped to the inlet face of CFD models using a temporallyvarying parabolic flow profile. Flow waveforms similarly obtainedfrom the head and neck arteries were used with measured BP data toprescribe outflow boundary conditions. Specifically, to replicate thephysiological effect of arterial networks downstream of the CFDmodel branches, three-element Windkessel model outlet boundaryconditions were imposed using a coupled-multidomain method (69).This method provides an intuitive representation of the arterial treebeyond model outlets and can be described by parameters withphysiological meaning that were calculated and iteratively adjusted aspreviously described (37, 47, 62) such that measured BP was repli-cated. Time-dependent CFD simulations were performed using anin-house stabilized finite-element solver with the embedded commer-cial linear solver LESLIB (Altair Engineering, Troy, MI) to solvetime-dependent Navier-Stokes equations. Vessels were modeled asrigid. Blood was assumed to be a Newtonian fluid with a density of1.06 g/cm3 and a viscosity of 4 cP consistent with a previous report(74) in rabbits and upon consideration of the shear rates observed inthe present investigation. Computational meshes contained �4 mil-

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lion tetrahedral elements, and localized refinement was performedusing an adaptive technique to deposit more elements in regions proneto flow disruption (45). Simulations were run for four to six cardiaccycles until the flow rate and BP fields yielded periodic solutions.Results for blood flow velocity, BP, and WSS were visualized usingParaView (Kitware, Clifton Park, NY). Time-averaged WSS (TAWSS)and the oscillatory shear index (OSI) were then calculated (39). LowTAWSS is thought to promote atherogenesis, as is elevated OSI, anindex of directional changes in WSS (39, 64). An OSI value of zeroindicates that WSS is unidirectional, whereas a value of 0.5 isindicative of bidirectional WSS with a time-averaged value of zero. Aprevious imaging study (25) found local low TAWSS and elevatedOSI values that were statistically different from circumferential aver-ages, motivating the need to report detailed local WSS results in CFDstudies. Therefore, circumferential values were extracted near loca-tions corresponding to where histological and myograph analyseswere conducted. Spatially equivalent regions were queried for allrabbits using the worst-case CoA rabbits as a guide. Thus, the regiondenoted as proximal represents the approximate midway locationbetween the left subclavian artery and coarctation, whereas the regiondenoted as distal is in the vicinity of the impact zone created by theimpinging velocity jet. Circumferential results at each location weredivided into 16 sectors of equal size, and values within each sectorwere averaged (36) to quantitatively determine the severity of local-ized hemodynamic alterations due to CoA and correction. Values forTAWSS were also extracted longitudinally along the anatomic rightand left luminal surfaces as well as the inner and outer curvatures ofthe thoracic aorta.

Histology and immunohistochemistry. Histology was conducted aspreviously described (43). Briefly, fixed arteries were dehydrated,embedded in paraffin wax, sectioned at 5 �m, and stained usingVerhoeff-Van Gieson methods to identify internal and external medialborders and elastic fibers for morphometric analysis of artery thick-ness. The medial area of arteries was also calculated by tracingborders of the internal elastic lamina (IEL) and external elastic laminaand subtracting the respective areas. This approach accounts forpossible differences in artery radius and thickness occurring due tofixation at varying BPs (75). All quantification was conducted intriplicate by three investigators blinded to the experimental group.

Expression of the cytosolic and extracellular focal adhesion pro-teins talin and fibronectin as well as the SM contractile differentiationmarker proteins SM �-actin, NM and SM myosin heavy chain, andcaldesmon were additionally evaluated using immunohistochemistry.Tissue specimens from the regions described above were cleaned ofblood and loose connective tissue, frozen in isopentane cooled inliquid nitrogen, and stored at �80°C. Sections (8 �m) were cut on aLeica CM1900 cryostat, mounted on glass slides, and stored at �20°Cuntil used. Tissues were then treated as previously reported (20).Briefly, frozen sections were fixed with 2% paraformaldehyde for 10min, permeabilized in 0.5% Triton X-100 for 10 min, and blockedwith 5 mg/ml BSA for 1 h. Sections were reacted with primaryantibody for 2 h and the appropriate secondary antibody for 2 h,incubated with 4=,6-diamidino-2-phenylindole (DAPI; 0.5 �M) tostain nuclei, and coverslipped. Multiple washes were used afterprimary and secondary incubation to avoid nonspecific binding. Allimmunoreacting solutions were made in PBS-Tween with 0.1% BSAas a carrier. Antibodies were obtained from the following sources: SM�-actin (A 2547), SM myosin (F0791), caldesmon (T3287), fibronec-tin (IST-3), and talin (8D4) from Sigma Chemical (St. Louis, MO),NM myosin (BT-561) from Biomedical Technologies (Stoughton,MA), Cy2 and Cy3 secondary antibodies from Jackson ImmunoResearch (West Grove, PA), and DAPI (157574) from MolecularProbes/Invitrogen (Eugene, OR).

Stained or immunoreacted sections were observed with an Olym-pus IX70 microscope using epifluorescense illumination where ap-propriate. To view the relatively large media of aortic sections, allimages were taken with a 10 air lens. Quantitative analysis was

performed using ImageJ. Digital grayscale images for SM �-actin,SM and NM myosin, caldesmon, fibronectin, and talin were collectedusing similar light intensity and exposure times for all samples with a16-bit Princeton Instruments camera controlled through a PCI boardvia IPLab for Windows. For each animal, three regions of interesthaving equal size and spanning the entire medial wall were randomlychosen. The mean optical density was then determined for eachlocation and averaged, with data expressed as average values ofstaining intensity. Four to five animals were quantified for eachexperimental group.

Myograph analysis. Vascular rings (width: 3–4 mm) were sec-tioned, cleaned of adhering perivascular tissue, mounted on an iso-metric myograph (Harvard Apparatus, Holliston, MA), maintained ina water-jacketed tissue bath in PSS bubbled continuously with O2 at37°C, and allowed to equilibrate for at least 1 h. An optimal restingforce of 2 g was applied to all sections based on preliminary exper-iments with this model (43, 58). Arteries underwent K-PSS contrac-tion followed by three PSS rinses, and this procedure was conductedtwo to three times until the effective force response of an artery wasconsistent (27, 59). To quantify endothelial function, aortic rings weresubsequently precontracted with 0.2 �M phenylephrine (PE), andendothelium-dependent NO relaxation was determined by cumulativeadditions of ACh (10�9–10�5 M) (3, 4). Samples were washed at leastthree times with fresh PSS and equilibrated for �1 h before endothe-lium-independent vasorelaxation to cumulative doses of sodium ni-troprusside (SNP; 10�9–10�5 M) was determined.

Aortic rings were then again allowed to equilibrate at resting forcebefore being stimulated with cumulative concentrations of PE (10�9–10�5 M). The effective force response to each PE concentration wasnormalized to the tissue’s maximum K-PSS contractile response,and cumulative dose-response curves were constructed.

Statistical analysis. All data are presented as means � SE. Statis-tical evaluation was performed using one-way ANOVA followed byTukey post hoc analysis. P values of �0.05 were considered signif-icant.

RESULTS

Maximum intensity projections (Fig. 1) of the acquiredMRA data confirmed that rabbits undergoing coarctation withsilk suture developed a pronounced stenosis after surgery,similar to untreated CoA in humans. Rabbits undergoing co-arctation with degradable Vicryl suture initially developedstenosis; however, complete degradation of the suture by56–70 days returned aortic diameter close to normal withmodest residual narrowing present in the suture region, asshown in Fig. 1. These morphological characteristics are sim-ilar to human surgical treatment of resection with end-to-endanastomosis, the most common method of surgical treatmentfor coarctation. Control rabbits represented healthy subjects ofsimilar age and weight.

BP. Rabbits undergoing coarctation with silk suture had asignificantly increased mean BP gradient of 20 � 2.0 mmHgacross the stenotic region compared with control rabbits (3.2 �1.7 mmHg) and corrected rabbits (2.7 � 1.3 mmHg, n � 7rabbits/group for all BP measurements). Representative wave-forms (data not shown) revealed increased proximal systolic,mean, and pulse BP but reduced distal pulse BP in CoA rabbits.Corrected rabbits had BP waveforms similar to control rabbits,with systolic, diastolic, and mean BP significantly less than theCoA group at the proximal location. Distal artery BP record-ings revealed no significant differences in systolic or mean BPacross all groups; however, pulse BP was significantly reducedin the CoA group compared with the control group. Values foreach group are shown in Table 1.

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CFD simulations. Computational simulation results for peaksystolic blood flow velocity patterns for all rabbits within eachexperimental group are shown in Fig. 2. Systolic velocityprofiles in control rabbits were generally parabolic, with peakvalues of �60 cm/s present throughout the aorta and branches.CoA rabbits showed lower velocity in the aortic arch, dramaticvelocity jet due to the coarctation, and a region of poststenoticdilation and tortuosity distal to the coarctation. Correctedrabbits demonstrated a region of increased velocity at thecoarctation site, where residual narrowing was present, andreduced velocity magnitude in the distal region, where amoderate dilation was present.

Control rabbits had fairly consistent distributions ofTAWSS, particularly in the dAo, ranging from 15–20 dyn/cm2

(Fig. 3). CoA rabbits showed marked differences, includingregions of low TAWSS proximal to the coarctation and ex-tremely high TAWSS at the location where the velocity jet dueto the coarctation impinged on the posterior portion of the dAodistally. The corrected group revealed a small region of in-creased TAWSS at the site of residual narrowing and reducedTAWSS distal to the coarctation.

Spatial distributions of OSI for all rabbits within eachexperimental group are shown in Fig. 4. Values generallyranged from 0.20 to 0.25 in the aortic arch and distal dAo ofcontrol rabbits. In contrast, CoA rabbits had elevated OSIimmediately distal to the coarctation and in the tortuous regionwith values of 0.40 to 0.50. These trends were similarly presentin corrected rabbits, but to a lesser extent.

Local TAWSS quantification. TAWSS results unwrappedabout the inner curvature of the dAo are shown in Fig. 5. In theregion of circumferential quantification proximal to the coarc-tation, CoA rabbits had significantly reduced TAWSS com-pared with control rabbits along the outer wall, but no differ-ences were observed for the corrected group. In the distalregion of quantification, CoA TAWSS was significantly ele-vated at the site where the velocity jet impinged in the outer leftluminal surface (100–130 dyn/cm2 greater than control rab-bits). In contrast, corrected rabbits showed significantly re-duced TAWSS in the inner right luminal surface distal to thecoarctation compared with control rabbits at this location(corrected rabbits: 3.3–5.4 dyn/cm2 vs. control rabbits: 13.8–17.5 dyn/cm2).

The longitudinal plots of TAWSS 1–10 diameters down-stream of the left subclavian artery shown in Fig. 5, right,further demonstrated the regional differences between groups.CoA rabbits showed drastically increased TAWSS (700–780dyn/cm2) at 2–2.5 diameters, corresponding to the locationwhere the lumen radius was reduced due to the suture. CoArabbits also displayed significantly reduced TAWSS proximalto the suture at 1.0–1.5 diameters along the right and outerluminal surfaces (CoA rabbits: 4.6–7.9 dyn/cm2 vs. controlrabbits: 15.4–31.7 dyn/cm2) as well as significantly reducedTAWSS from �3–6 diameters along the right, outer, and leftluminal surfaces (CoA rabbits: 1.1–8.7 dyn/cm2 vs. controlrabbits: 12.4–21.4 dyn/cm2). The stenotic velocity jet resultingfrom the coarctation caused increased TAWSS in a rotationalmanner at 5.5–7 diameters along the outer luminal surface,6.5–8 diameters along the left luminal surface, and 8–9.5diameters along the inner luminal surface (CoA rabbits: 110.9–136.6 dyn/cm2 vs. control rabbits: 18.5–21.1 dyn/cm2). Cor-

CONTROL CoA CORRECTED

Fig. 1. Representative maximum intensity projections of magnetic resonance angiography data from each experimental group. All images were obtained at theconclusion of the experiment, 1 day before the completion of the experiment at 32 wk of age. For corrected and coarctation of the aorta (CoA) groups, this was�22 wk after the coarctation was induced.

Table 1. BP measurements obtained at carotid (proximal)and femoral (distal) locations

Groups

Control CoA Corrected

ProximalSystolic BP 71 � 3 99 � 7*† 69 � 3Mean BP 64 � 4 87 � 8*† 61 � 4Diastolic BP 58 � 4 74 � 10 54 � 4Pulse Pressure 13 � 1 25 � 4*† 16 � 1

DistalSystolic BP 78 � 5 70 � 9 73 � 4Mean BP 61 � 4 67 � 9 58 � 4Diastolic BP 52 � 4 64 � 8 50 � 4Pulse Pressure 25 � 1 6 � 2*† 22 � 1

Values are means � SE; n � 7 rabbits/group. BP, blood pressure. *Coarc-tation of aorta (CoA) group significantly different the control group (P �0.05); †CoA group significantly different from the corrected group (P � 0.05).



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rected rabbits also showed a reduction in TAWSS proximal tothe suture at 1.0–1.5 diameters in the outer luminal surface(corrected rabbits: 13.9–16.2 dyn/cm2 vs. control rabbits:29.0–31.7 dyn/cm2), similar to the CoA group, as well asreduced TAWSS in the right, outer, and left luminal surfaces at3.5–4.5 diameters. Corrected rabbits also revealed reducedTAWSS along the inner and right luminal surfaces from5.5–7.5 diameters, in agreement with circumferential data, andat 9.5–10 diameters in the outer luminal surface.

Histology and immunohistochemistry. Figure 6 shows rep-resentative Verhoeff-Van Gieson-stained images at each re-gion, where the hatched portions of the bars in Fig. 6, right,indicate the amount of medial thickness containing disorga-nized or fragmented elastic lamellae not present in the controlgroup. In both left common carotid artery and proximal dAoregions, total medial thickness was significantly increased withpronounced elastin fragmentation in the CoA and correctedgroups compared with the control group. These differences inmedial thickness were not present distal to the coarctation site.Similarly, medial area quantified in the proximal dAo wassignificantly greater for CoA and corrected rabbits comparedwith control rabbits, but there were no differences betweengroups in the distal dAo (proximal dAo: 2.02 � 0.15 mm2 incontrol rabbits, 5.70 � 0.74 mm2 in CoA rabbits, and 3.96 �0.18 mm2 in correct rabbits; distal dAo: 1.97 � 0.07 mm2 incontrol rabbits, 2.37 � 0.15 mm2 in CoA rabbits, and 2.41 �0.23 mm2 in corrected rabbits).

NM myosin staining intensity was significantly increased inproximal dAo arteries of both CoA and corrected rabbits com-pared with control rabbits (CoA and corrected rabbits: 87.0 �6.4 and 83.2 � 3.6, respectively, vs. control rabbits: 37.6 �2.6; Fig. 7). These differences were not present in the distaldAo region. No significant differences were observed in fluo-rophore intensity radially across the media. The use of histo-gram distributions to further detect any spatial trends in thedistribution of NM myosin also did not yield any significantdifferences. SM myosin staining intensity of proximal arterieswas significantly reduced in CoA and corrected groups com-pared with the control group (CoA and corrected groups: 51.6 �4.4 and 57.7 � 5.8, respectively, vs. control group: 118 � 11.2),whereas no significant differences in SM myosin intensity werepresent distally. Caldesmon (data not shown) intensity was sig-nificantly reduced in the proximal arteries of CoA rabbits (51.5 �4.0 in CoA rabbits vs. 116 � 6.3 in control rabbits), whereas nodifferences were present distally. There were also no statisticaldifferences in talin, fibronectin (data not shown), or SM �-actinstaining intensity across groups.

Myograph analysis. Arterial relaxation curves in response tothe endothelium-dependent agonist ACh and the endothelium-independent agonist SNP are shown in Fig. 8 for proximal anddistal locations. Proximal to the coarctation, control rabbitsshowed intact endothelial function by demonstrating AChrelaxation to �80% of precontracted force. In contrast, bothCoA and corrected rabbits showed significantly impaired ACh

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Fig. 2. Volume-rendered peak blood flow velocity patterns for 32-wk-old male New Zealand White rabbits. Results from control rabbits (top) are shown relativeto those from rabbits that underwent aortic coarctation using permanent (CoA; middle) or degradable (corrected; bottom) sutures at 10–12 wk of age. Eachnumber (1–7) refers to an individual rabbit, and the numbering of rabbits is consistent in Figs. 3–5.



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relaxation (e.g., �3 � 11% and 27 � 9%, respectively), withCoA rabbits showing marked impairment at peak ACh values.In the distal region, differences between corrected and controlgroups were absent, with relaxations of 86 � 2% and 78 � 3%,respectively. In contrast, CoA rabbits continued to show sig-nificantly impaired ACh relaxation distally, with a peak relax-ation of �18 � 11% of precontracted force.

Endothelium-independent relaxation in the proximal dAoreached �90% of precontracted values for control rabbits. Asmall but significant reduction in relaxation for CoA comparedwith control rabbits was present at intermediate doses of SNP[log(�7.5 to �6.5)]; however, peak SNP relaxation was notsignificantly different between all groups. Corrected rabbitsshowed similar SNP relaxation responses compared with con-trol rabbits regardless of dose. In the distal dAo, corrected andcontrol rabbits continued to show similar SNP relaxation re-sponses, whereas CoA rabbits had significantly greater relax-ation compared with the corrected group at intermediate doses[log(�7 to �4.5)] and control rabbits at peak doses (98 � 1%in CoA rabbits vs. 90 � 1% in control rabbits).

The PE force response at proximal and distal dAo locationsis also shown in Fig. 8. In the proximal dAo, control rabbitsshowed a normalized effective force response peaking at1.13 � 0.08, whereas both CoA and corrected rabbits peakedat only 0.94 � 0.04 and 0.92 � 0.03, respectively, and showed

significantly diminished force at several concentrations. PeakK-PSS contraction results are shown in Table 2, and proximalaortic rings demonstrated significantly reduced force in cor-rected and CoA rabbits compared with control rabbits, similarto PE contraction trends. In the distal dAo, control rabbitsdemonstrated a peak contractility of 1.04 � 0.05, and the CoAforce response was statistically unchanged throughout. Inter-estingly, the corrected force response remained significantlyreduced compared with control rabbits, with a peak effectiveresponse of only 0.88 � 0.06. K-PSS force from distal aorticrings was similar between corrected and control groups,whereas K-PSS force in the CoA group was significantlyincreased (Table 2).

Cyclic strain. CoA and corrected rabbits demonstrated sig-nificantly reduced mean and maximum strain as well as dis-tensibility compared with control rabbits, with CoA rabbitsshowing the greatest reductions compared with corrected rab-bits (Table 3). The pressure-strain elastic modulus was signif-icantly increased in CoA rabbits (47.2 � 4.66 103 N/m2)compared with both control (8.46 � 1.05 103 N/m2) andcorrected (15.6 � 1.39 103 N/m2) rabbits, indicatingincreased stiffness in CoA rabbits. Ascending aortic diam-eter in CoA rabbits was significantly greater than in controlrabbits: 8.98 � 0.39 vs. 7.53 � 0.23 mm. Corrected rabbits

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Fig. 3. Spatial distributions of time-averaged wall shear stress (TAWSS) for 32-wk-old male New Zealand White rabbits. Results from control rabbits (top) areshown relative to CoA (middle) or corrected (bottom) rabbits at 10–12 wk of age. Each number (1–7) refers to an individual rabbit, and the numbering of rabbitsis consistent in Figs. 2, 4, and 5. Arrows on the models in rabbit 1 indicate where circumferential TAWSS was obtained for the plots shown in Fig. 5.



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showed a slight increase in mean diameter (8.14 � 0.20 mm),but this did not reach significance.

DISCUSSION

The objective of this work was to use an experimental modelof CoA and correction to quantify alterations in hemodynamicand vascular biomechanics indexes and the associated expres-sion of key SM contractile and focal adhesion proteins alongwith their relationship to vascular remodeling, endothelialdysfunction, and stiffness. There were three important findings,as discussed in more detail below: 1) in addition to themechanical stimuli of elevated BP, CoA induces significantalterations in WSS that persist despite the total alleviation ofthe BP gradient across the coarctation; 2) endothelial dysfunc-tion exists in both CoA and corrected rabbits; and 3) proximalNM myosin expression is increased and SM myosin expressionis decreased in both CoA and corrected rabbits at locationswhere there is vascular remodeling with altered function andstiffness.

TAWSS alterations in untreated and corrected CoA. Thepresent results confirm and extend preliminarily results docu-mented in our previous work (43) by showing that significantalterations in TAWSS occur in both untreated and correctedCoA using a novel rabbit model. Low TAWSS occurs in arotating pattern down the dAo of healthy adults (25, 32), and astudy (73) has shown a correlation in these sites with athero-

sclerotic plaques. In the present investigation, CFD was used toquantify local hemodynamics circumferentially and longitudi-nally in untreated and corrected CoA rabbits compared withcontrol rabbits using MRI and BP data. Untreated CoA rabbitsdemonstrated reduced TAWSS proximal to the coarctation andsignificantly elevated TAWSS distally due to the velocity jet.Results from the corrected rabbits demonstrated reducedTAWSS proximal to the suture and furthermore that the ve-locity jet may have persistent effects on tortuosity, with re-duced TAWSS occurring in a rotational pattern along the distalwall due to the influence of the jet. These alterations toTAWSS caused by CoA that persist upon correction may thusindicate an increased risk of atherosclerotic plaque formationin the dAo of these subjects. While the general patterns of WSSand OSI in this study correspond well with available data inhumans (35, 36), the use of a representative rabbit modelallows the observation of CoA-induced alterations independentof any confounding factors or heterogeneity often present inclinical populations.

Endothelial dysfunction in untreated and corrected CoA.Proximal dAo arteries from untreated and corrected rabbitsexhibited endothelial dysfunction, as ACh relaxation curvesfrom both groups showed significant impairments, whereas thepeak SNP relaxation response (endothelium independent) wasunchanged. Distal arteries of the CoA group continued to showendothelial dysfunction, but no differences between corrected

CO

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Fig. 4. Spatial distributions of the oscillatory shear index for 32-wk-old male New Zealand White rabbits. Results from control rabbits (top) are shown relativeto CoA (middle) or corrected (bottom) rabbits at 10–12 wk of age. Each number (1–7) refers to an individual rabbit, and the numbering of rabbits is consistentin Figs. 2, 3, and 5.



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and control groups were present. While endothelial dysfunc-tion in the proximal arteries of untreated CoA rabbits is inagreement with spontaneously hypertensive animal models(16, 28, 38), the data presented from corrected rabbits are thefirst to demonstrate endothelial dysfunction in coarctationdespite the restoration of normal BP using a chronic animalmodel mimicking treatment. Importantly, researchers investi-

gating tissue specimens for viability often consider rabbitarteries exhibiting �50% relaxation to be nonfunctional (41),and maximum ACh relaxation from CoA and corrected rabbitsin the present study was 17% and 32%, respectively, under-scoring the severity of this dysfunction. These results areconsistent with recent clinical evaluations of normotensivepostrepair patients who demonstrated reduced forearm vasodi-

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Fig. 5. Left: local quantification conducted using unwrapped TAWSS results. Numbers (1–7) next to unwrapped images refer to an individual rabbit, and thenumbering of rabbits is consistent in Figs. 2–4. The locations of circumferential quantification are indicated by dashed lines and correspond to arrows on modelsin rabbit 1 in Fig. 3. These locations represent regions in the proximal and distal descending thoracic aorta where histological and myograph arteries wereobtained. Middle: the two images show the convention used to define luminal surfaces by their outer or inner curvatures (top image) and anatomic left or rightluminal surfaces (bottom image) and provide a key for the division of circumferential TAWSS plots into 16 equal sectors along these surfaces. The plots showensemble-averaged circumferential TAWSS results (proximal and distal plots), with the distal TAWSS plot additionally magnified to elucidate differencesbetween control and corrected values (distal zoomed plot). Right: ensemble-averaged longitudinal TAWSS plots along the outer, anatomic right, anatomic left,and inner luminal surfaces for the collection of rabbits in each group. Please note the breaks in the ordinate axes of longitudinal plots that were necessary toaccommodate elevated TAWSS values in the coarctation. *CoA different from control rabbits; †corrected rabbits different from control rabbits; §CoA rabbitsdifferent from corrected rabbits (all P � 0.05).



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lation during reactive hyperemia compared with age-matchedcontrol patients (11, 12). Atherosclerosis, particularly in coro-nary artery disease, is associated with endothelial dysfunctionas a result of reduced endothelial NO release (17, 18, 66), and,indeed, high rates of early-onset coronary artery disease are aprimary form of morbidity leading to reduced life expectancyin surgically treated CoA. Whether the etiology of endothelialdysfunction observed here is a result of ROS or increasedexpression of inflammatory or adhesion molecules is currentlyunclear, as studies have reported evidence of both phenomena(5, 12, 67).

The finding of endothelial impairment in distal arteries ofCoA rabbits may be attributable to the presence of highvelocity and WSS in these regions. Previous research (42) hasobserved remodeling and fragmentation of the IEL underincreased blood flow rates. Thus, sustained conditions of in-

creased WSS in the CoA group may serve to alter the endo-thelial environment and subsequently NO release. IEL frag-mentation due to high WSS may also explain the slight in-crease in the distal dAo SNP relaxation response at severalconcentrations, as NO may diffuse more readily into medialSM to cause relaxation.

Altered protein expression occurs with vascular remodeling,aortic stiffening, and reduced active contractile responses.Analysis of phenotypic SM modulation, manifested by immu-noreactivity of contractile and focal adhesion proteins, re-vealed increased NM myosin and decreased SM myosin ex-pression in CoA and corrected rabbits proximally, whereas SM�-actin was unchanged. Phenotypic modulation occurring viadecreased SM �-actin has been reported in several studies ofhypertension and atherosclerosis (13, 52). However, few ofthese studies to date have observed the SM response in the

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Fig. 6. Verhoeff-Van Gieson-stained arterial sections representative of sections obtained proximal (carotid) and distal (femoral) to the coarctation region in CoAand corrected groups compared with spatially equivalent locations from the control group. Hatched portions of the plots on the right indicate the amount of themedial layer containing fragmented lamellae devoid of darkly stained elastin, as shown by the line placed orthogonally on the proximal CoA section. Upwarderror bars on the plots correspond to means � SE for the entire medial thickness, while downward error bars represent means � SE for hatched portions of theplots. *Significantly different from the control group (P � 0.05).



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Fig. 7. Left: representative micrographs of immunohistochemical staining of the proximal (top) and distal (bottom) aorta with nonmuscle (NM) myosin, smoothmuscle (SM) �-actin, and SM myosin. Right: quantified staining intensity (means � SE, pixel counts) for each location and group. *Significantly different fromthe control group (P � 0.05).



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large arteries, and previous work with animal models of co-arctation has demonstrated altered expression of SM contrac-tile proteins without significant depression of SM �-actin (29,31). It was reasoned in these previous studies that medial SMmay be “multifunctional” and that loss of SM �-actin is not anabsolute prerequisite for phenotypic changes. No changes wereobserved in the focal adhesion-associated proteins fibronectinand talin, suggesting that these extracellular and cytoskeletal(adherens juctions) proteins may not play a key role in remod-eling in response to CoA.

Previous work has shown NM myosin to have mechanosen-sitive properties that allow it to respond to changes in mechan-ical stimuli through its expression and activation. Increasedapplication of cyclic strain reduces NM myosin expression(54), and more recent studies (6, 7) have demonstrated NMmyosin activation to be increased in cell cultures with greaterrigidity. The mechanosensitive response of NM myosin isthought to involve changes to its actin-linking and contractilefunctions, which subsequently affect SM cell migration andadhesion. In the present study, mechanical stimuli in the formof increased systolic, mean, and pulse BP appear to result inincreased NM myosin expression with decreased SM myosin

and thus a shift in phenotype from contractile to synthetic SMisoform expression. This dedifferentiation may lead to medialthickening and elastin fragmentation at locations near the siteof coarctation (proximal dAo). This response appears to be partof a compensatory mechanism to restore tensile stress tohomeostatic levels by increasing wall thickness and may occurthrough SM proliferation, hypertrophy, and increased extracel-lular matrix deposition. Previous models of coarctation-in-duced hypertension have demonstrated vascular remodeling tooccur as far upstream as the coronary arteries but not thecerebral vasculature (60). In the present study, medial thick-ening was observed upstream of the CoA in the carotid arteries,but immunohistochemistry data were not available to link thesestructural changes with SM phenotypic alterations. Medialthickening in the corrected group despite BP alleviation is ofparticular interest as it demonstrates the establishment of vas-cular remodeling processes that persist after the removal of themechanical stimuli and is in agreement with postrepair clinical

Table 3. Strain parameters delineated from phase-contrastMRI imaging data

Group


Mean E�� 0.138 � 0.013 0.044 � 0.004*‡ 0.068 � 0.004†Maximum E�� 0.274 � 0.025 0.082 � 0.005*‡ 0.156 � 0.003†Elastic modulus, N/m2 8.46 � 1.05e3 47.2 � 4.66e3*‡ 15.6 � 1.39e3

Mean diameter, mm 7.531 � 0.225 8.976 � 0.393* 8.137 � 0.195Aortic distensibility 0.038 � 0.005 0.008 � 0.002*‡ 0.019 � 0.002†

Values are means � SE; n � 7 rabbits/group. E��, Green-LaGrange strain.*CoA group significantly different from the control group (P � 0.05);†corrected group significantly different from the control group (P � 0.05);‡CoA group significantly different from the corrected group (P � 0.05).

-9 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.50.0

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Fig. 8. Active vasorelaxation curves in response to ACh (left) and sodium nitroprusside (middle) and contractile curves in response to phenylephrine (right) forrings of aortic tissue extracted proximal and distal to the coarctation region in CoA and corrected groups compared with spatially equivalent locations from thecontrol group. *CoA rabbits significantly different from control rabbits (P � 0.05); †corrected rabbits significantly different from control rabbits (P � 0.05);§CoA rabbits significantly different from correct rabbits (P � 0.05).

Table 2. Peak K-physiological salt solution contractions

Groups


Proximal 13.31 � 1.12 8.27 � 0.36* 8.28 � 0.14†Distal 11.05 � 1.19 17.03 � 1.09*‡ 11.73 � 0.46

Values are means � SE; n � 4 rabbits/group. *CoA group significantlydifferent from the control group (P � 0.05); †corrected group significantlydifferent from the control group (P � 0.05); ‡CoA group significantly differentfrom the corrected group (P � 0.05).



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reports (71, 72) of increased carotid intima-media thickness inhuman patients.

Vascular remodeling due to NM and SM myosin expressionchanges may cause proximal arterial stiffening, manifestedthrough indexes of reduced distensibility and strain in untreatedand corrected CoA. While strain indexes in the untreated groupare also likely due to the downstream coarctation, the findingsof reduced strain and distensibility in the corrected groupindicates that resting mechanical properties are altered despiteelevated BP alleviation and is consistent with a previous study(50) suggesting that altered vascular properties persist despitetreatment. Changes in resting mechanical properties are typi-cally reflected by alterations to collagen, elastin, and medialthickness (75), and aortic stiffening causes increased cardiacafterload and decreased coronary perfusion (46). The presentresults indicate that significant vascular SM dedifferentiation ispresent in these regions, and thus persistent vascular alterationsmay be caused by persistent vascular remodeling and extracel-lular matrix deposition, which increase medial thickness at theexpense of arterial stiffening.

Myograph results demonstrated reduced active contractiletone through diminished PE force response in proximal arteriesof both untreated and corrected rabbits. These findings aresomewhat paradoxical as vascular remodeling observed inthese proximal arteries would be expected to result in increasedvessel contractility (increased contractile units in parallel in-crease force); however, these findings may be attributable tothe shift in SM phenotype from contractile to synthetic, asevidenced by NM and SM myosin, which may cause theincrease resting properties and stiffness observed. It appearsthat the shift of SM to NM myosin has a larger effect on forcethan the increase in wall thickness, as average PE forcedecreases. To maintain the vessel diameter, previous work (10)using hypertensive rat arterioles has shown that increasingresting force decreases the capacity for an active force re-sponse. Although studied less frequently than resistance ves-sels, elastic conduit arteries demonstrate an ability to adapt SMtone in response to WSS and BP to control luminal diameter.It is possible that if NM and SM myosin expression changesand vascular remodeling increase wall stiffness in response toCoA-induced mechanical stimuli, active tension of proximalaortas in untreated and treated groups must be limited to avoidexcessive vasoconstriction. Since maximum active tension inthe CoA and corrected groups was reduced compared with thecontrol group in response to PE and K-PSS, it is likely thatactive tension is reduced in the presence of increased aorticstiffness and thus does not contribute to increased total force.In the distal aorta, a reduced active force response occurred incorrected rabbits without a transient increase in BP or myosinexpression changes and is thus not strictly consistent withproximal results. Since K-PSS force generated in distal aorticrings did not change significantly from that in the controlgroup, we hypothesize that the decreased PE response is notdue to a decreased ability of the SM to contract. The presenceof significantly reduced TAWSS in corrected rabbits at thisregion from CFD results may explain this reduced force, assignificant alterations in WSS may affect SM contractility (asdiscussed below). The fact that K-PSS contraction in distalCoA aortic rings was significantly greater than both control andcorrected aortic rings at this region may be related to the factthat NO relaxation in the CoA distal aorta was significantly

impaired. Since K-PSS force results from both corrected andCoA distal aortic rings were not consistent with the trendsobserved in PE force results at this region, there may be acombination of factors that contribute to these changes, andfurther studies will be required to fully address these phenom-ena.

The present results should be interpreted within the con-straints of several potential limitations. This work focused onthe expression of several key SM contractile and focal adhe-sion proteins associated with coarctation-induced vascular re-modeling and functional impairment. Previous work (13) hasdemonstrated that fibronectin plays a regulatory role in flow-induced vascular remodeling, and it is thus somewhat surpris-ing that spatial locations of reduced TAWSS, such as theproximal dAo in CoA and distal dAo in corrected rabbits, didnot show increased expression of fibronectin. This is perhapsexplained by the degree to which WSS was reduced by ligationin these prior experiments (�25 dyn/cm2 decrease), whereasthe present study observed relative reductions in TAWSS of�10–13 dyn/cm2.

Observed differences in active force across experimentalgroups are thought to be a result of changes in resting andactive components of SM contraction, independent of intracel-lular Ca2 regulation. However, it is conceivable that obser-vations may be a result of Ca2-dependent pathways, includingcGMP kinase and L-type channels, or Ca2-independent Gprotein coupling, all of which serve to alter contractility inresponse to sustained hemodynamic stimuli (1, 2, 14, 56).Future studies using inhibitors for Ca2 channels (verapamil)and agonist activation (phentolamine) are feasible using thecurrent methods and will allow the determination of Ca2-dependent regulation in contractile force response in untreatedand corrected CoA.

The mean Reynold’s number in the ascending aorta of therabbits in the present study ranged from �260–300, whereas aprevious study (36) investigating CoA in humans found a meanReynold’s number of �1,100–1,500. While flow may begenerally laminar in both cases, these differences suggest theflow regime of rabbits is viscous dominated, unlike the iner-tially dominated flow present in humans. An important impli-cation of these differing regimes is that turbulence and second-ary flows are not likely to occur in the rabbit aorta.

Simulations were performed using a rigid wall assumptionsince the version of CFD software used does not account forvariable compliance in the thoracic aorta present under controlor CoA conditions, detailed local material properties were notquantified, and to reduce computational expense. We (35) havepreviously observed that cyclic strain is reduced in treated CoAcompared with control patients, a finding also represented withthe present rabbit model of CoA. Hence, while the incorpora-tion of variable local vessel compliance could provide morerealistic values for indexes of WSS, it is unlikely that deform-able CFD results would lead to appreciable differences in thefindings presented here given the elevated stiffness observed inCoA and corrected rabbits.

Analysis of CFD model morphology shows that pronouncedtortuosity can develop distal to the coarctation. Anecdotally,we have noticed that the severity of this tortuosity appears to berelated to the location within the proximal aorta where the CoAis induced and whether a resulting velocity jet directly impactsthe distal posterior luminal surface and is then redirected down



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the dAo or is dissipated within the center of the downstreamaortic flow domain. Interestingly, this tortuosity is not typicallyobserved in humans with CoA. Turbulence has previously beenlinked to poststenotic dilation (34, 49, 76). Hence, it is possiblethat the viscous-dominated regime in the thoracic aorta of therabbit may limit poststenotic dilation that could occur inhumans and ameliorate the stimulus for tortuosity introducedby a high velocity jet. However, this hypothesis remains to betested.

Product literature for the Vicryl suture used with correctedrabbits (Polyglactin 910, Ethicon Novartis) indicates that thissuture is completely absorbed after 8–10 wk. While the exacttiming of when the coarctation is alleviated in vivo was notprecisely determined, the product literature provides an initialstrength of �15 lbf and a known strength retention curve basedon the number of days since suture implementation. An anal-ysis using this retention curve with our previous CFD resultsand knowledge of rabbit aortic diameters indicates that theforce exerted on the suture used to create the coarctation byintegrating the distribution of tractions within this region (22)exceeds the strength indicated by the manufacturer after �21days. Importantly, this information suggests that the approachof inducing CoA with dissolvable Vicryl suture used for thepresent investigation provides the stimulus of altered hemody-namic from CoA for 3 wk before restoring BP to normal for�4 mo (�6 human years) before the experimental end point.

Conclusions. This study is the first to provide a possibleexplanation involving persistent endothelial dysfunction andphenotypic changes in vascular SM via altered protein expres-sion as mechanisms for cardiovascular disease. These changesmay be useful as surrogate end points for subsequent cardio-vascular disease as they remain altered despite successfulrepair of aortic coarctation.

CoA appears to cause increased BP and tensile stress as wellas reduced TAWSS in proximal arteries, which results in SMCphenotypic changes, as evidenced by altered NM and SMmyosin expression. This dedifferentiation may result in in-creased medial thickness and stiffness, which reduces the needfor an active force response. Furthermore, the increased tensilestress and reduced TAWSS cause proximal endothelial dys-function in CoA. Distally TAWSS is markedly increased bythe stenotic velocity jet, which causes endothelial dysfunctionand increased SM (endothelium independent) relaxation. Theseresults are in partial agreement with previous evidence show-ing that high WSS results in fragmentation and disruptionsof the IEL, which may also explain altered mechanicalfunction (42).

Despite almost complete reversal of the stimuli for vascularalterations and CoA-induced morbidity in corrected rabbits (nochange in BP or TAWSS in the proximal arteries), SM celldedifferentiation persists and is coincident with increased me-dial thickness and increased wall stiffness, reducing the needfor active force response. Additionally, endothelial dysfunctionpersists in proximal corrected arteries and appears to be estab-lished within 3 wk of CoA. Distally, the stentoic velocity jetcauses a residual dilatation in the corrected rabbit aorta, whichresults in reduced TAWSS leading to a reduced SM cellcontractile response.

Taken together, these results provided further evidence thatthe ramifications of CoA go far beyond removal of the stenosisand restoration of a favorable BP gradient, as hemodynamic

indexes of morbidity persist under these current treatmentguidelines. Considering these alterations persist upon correc-tion, it is likely that current interventions for CoA could benefitby addressing these phenomena. Specifically, the data in thepresent study point to the possibility of inhibitors in the NMmyosin activation pathway that may have therapeutic applica-tions in CoA.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Eric Jensen (Biomedical ResourceCenter, Medical College of Wisconsin), Mia Helfrich (Biomedical Engineer-ing, Brown University), Paul Larsen (Tulane University School of Medicine),Ronak Dholakia (Cerebrovascular Center, Stony Brook University MedicalCenter), and John Tessmer and David Schwabe (Department of Anesthesiol-ogy, Medical College of Wisconsin) for experimental assistance. The authorsalso thank Tina Kostenko, Yu Liu, Eric Paulson, and Julie Peay (Departmentof Biophysics, Medical College of Wisconsin) for imaging assistance andChristy Stadig and Wale Sulaiman (Department of Neuroscience/NeuroscienceResearch Labs, Medical College of Wisconsin) for the use of monitoringequipment during imaging sessions.

GRANTS

This work was supported by National Heart, Lung, and Blood InstituteGrant R15-HL-096096-01 (to J. F. LaDisa, Jr.) and assistance from the Alvinand Marion Birnschein Foundation (to J. F. LaDisa, Jr.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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