endothelial nitric oxide synthase activity is essential...

Endothelial Nitric Oxide Synthase Activity Is Essential forVasodilation During Blood Flow Recovery but not

for ArteriogenesisBarend Mees, Shawn Wagner, Elena Ninci, Silvia Tribulova, Sandra Martin, Rien van Haperen,

Sawa Kostin, Matthias Heil, Rini de Crom, Wolfgang Schaper

Objective—Arteriogenesis is the major mechanism of vascular growth, which is able to compensate for blood flowdeficiency after arterial occlusion. Endothelial nitric oxide synthase (eNOS) activity is essential for neovascularization,however its specific role in arteriogenesis remains unclear. We studied the role of eNOS in arteriogenesis using 3 mousestrains with different eNOS expression.

Methods and Results—Distal femoral artery ligation was performed in eNOS-overexpressing mice (eNOStg), eNOS-deficient (eNOS�/�) mice, and wild type (WT) controls. Tissue perfusion and collateral-dependent blood flow weresignificantly increased in eNOStg mice compared with WT only immediately after ligation. In eNOS�/� mice, althoughtissue perfusion remained significantly decreased, collateral-dependent blood flow was only decreased until day 7,suggesting normal, perhaps delayed collateral growth. Histology confirmed no differences in collateral arteries ofeNOStg, eNOS�/�, and WT mice at 1 and 3 weeks. Administration of an NO donor induced vasodilation in collateralarteries of eNOS�/� mice, but not in WT, identifying the inability to vasodilate collateral arteries as main cause ofimpaired blood flow recovery in eNOS�/� mice.

Conclusions—This study demonstrates that eNOS activity is crucial for NO-mediated vasodilation of peripheral collateralvessels after arterial occlusion but not for collateral artery growth. (Arterioscler Thromb Vasc Biol.2007;27:1926-1933.)

Key Words: endothelial nitric oxide synthase � arteriogenesis � mouse � hind limb � vasodilation

The stimulation of vascular growth has become an impor-tant therapeutic goal for prevention and treatment of

tissue ischemia in cardiovascular disease and is referred to astherapeutic neovascularization. Three distinct processes ofvascular growth can contribute to the recovery of blood flowand preservation of tissue: arteriogenesis, ie, collateral arterygrowth, angiogenesis, ie, sprouting of capillaries from preex-isting blood vessels, and vasculogenesis, ie, formation ofblood vessels from endothelial progenitors.1 From a thera-peutic point of view, it is essential to isolate the distinctmechanisms of vascular growth, because these occur indifferent types of tissue and vessels and are regulated byseparate stimuli. For example, in the experimental ischemichind limb model, arteriogenesis is initiated and stimulated incollateral vessels in the upper part of the hind limb by anincrease in fluid shear stress.2 In contrast, in the lower part ofthe limb, both angiogenesis and vasculogenesis are mainlydriven by tissue ischemia.3,4 Clinical trials based on stimula-tion of therapeutic VEGF- and FGF-mediated angiogenesis

have not shown convincing results.5,6 Therefore, in this studywe focus on arteriogenesis, as this is the most upstreammechanism and the most efficient one to provide bulk flow tothe ischemic area after occlusion or stenosis of a majorartery.7

A potential mechanism for inducing therapeutic neovascu-larization is to increase the production of endothelial NO.eNOS activity has been shown essential for neovasculariza-tion. eNOS�/� mice display a decreased neovascularization inresponse to severe ischemia. Recent studies have attributedthis to impaired arteriogenesis, angiogenesis, vasculogenesis,or a combination of these.8–10 However, in these studies thedifferent competing or complementary mechanisms of vas-cular growth could not be isolated because of the use of asevere murine ischemia in vivo model, which causes substan-tial damage to the lower limb. Thus, the role of eNOS in eachspecific mechanism could not be elucidated. In addition, therole of eNOS in arteriogenesis has been paradoxical. In-creased shear stress is known to upregulate the expression of

Original received May 30, 2006; final version accepted April 20, 2007.From the Department of Experimental Cardiology (S.W., E.N., S.T., S.M., S.K., M.H., W.S.), Max-Planck-Institute for Heart & Lung Research, Bad

Nauheim, Germany; and the Departments of Cell Biology & Genetics (B.M., R.v.H., R.d.C.) and Vascular Surgery (B.M., R.d.C.), Erasmus UniversityMC, Rotterdam, The Netherlands.

Correspondence to Barend M.E. Mees, MD, Department of Cell Biology & Genetics, Erasmus University MC Rotterdam, Dr. Molewaterplein 50, 3015GE Rotterdam, The Netherlands. E-mail [email protected]

© 2007 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.107.145375

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eNOS augmenting endothelial NO production.11–13 In con-trast, NO has also been shown to inhibit expression ofadhesion molecules and smooth muscle cell proliferation,which are both indispensable for arteriogenesis.11,14,15 Be-sides, given the importance of eNOS in regulating vasculartonus and blood flow, other mechanisms could be involved.

In this study, we investigated the role of eNOS in arterio-genesis in both eNOS�/� and eNOStg mice using a hind limbmodel, which caused only minimal ischemia in the lowerlimb, to specifically analyze arteriogenesis. Using this murinearteriogenesis-specific model several research groups haverecently described different molecular and cellular mecha-nisms of arteriogenesis.16–18 Two lines of eNOStg mice havebeen previously generated in our laboratory and overexpressthe human eNOS gene.19,20 We have recently reported that inthe eNOStg mice eNOS expression is functional and re-stricted to the endothelial lining in all blood vessels. IneNOStg mice eNOS protein and eNOS activity (20-foldgreater) levels in the vasculature as well as NO-production(1.8-fold increased) are significantly enhanced, causing alower blood pressure, lower plasma cholesterol levels, andless atherosclerosis.

In the present study, we first compared eNOStg and WTmice and only found beneficial effects of eNOS overexpres-sion on blood flow recovery immediately after ligation and nofurther favorable effects on collateral artery growth. Subse-quently, we analyzed arteriogenesis in eNOS�/� and WTcontrol mice and confirmed previously published data show-ing impaired blood flow recovery and increased ischemictissue damage in eNOS�/� mice. However, we found nodifferences in collateral growth between eNOS�/� and WTmice. Interestingly, we discovered that in eNOS�/� miceblood flow recovery and clinical outcome after distal femoralligation were impaired by the inability to sufficiently vaso-dilate collateral peripheral vessels, and not because of im-paired arteriogenesis.

Materials and MethodsAnimal ExperimentsTransgenic mice overexpressing eNOS-GFP (eNOStg) were gener-ated as previously described.20 Briefly, an eNOS-GFP fusion genewas made by inserting a DNA fragment encoding the enhanced greenfluorescent protein (GFP) in frame at the stop codon of the completehuman eNOS gene and used to perform microinjections of fertilizedmouse oocytes. Hemizygous eNOStg mice were used that expressedthe human eNOS gene fused to GFP under the regulation of theautologous human eNOS promoter and that were backcrossed toC57BL/6J background for �10 generations. For further details, seesupplemental methods, available online at http://atvb.ahajournals.org. eNOS deficient (eNOS�/�) and littermate wild type C57Bl/6mice (WT) were originally purchased from Jackson Laboratories(Bar Harbor, Maine). All mice used were age- (10 to 12 weeks) andsex-matched. The experimental protocol was approved by the Ani-mal Experiments Committee under the National Experiments onAnimals Act and adhered to the rules laid down in this national lawthat serves the implementation of “Guidelines on the protection ofexperimental animals ” by the Council of Europe (1986), Directive86/609/EC.

Mouse Model of Femoral LigationThe surgical procedure was performed as previously described.7

Briefly, mice were anesthetized with a mixture of ketamin (20

mg/kg) and xylazin (110 mg/kg). After minimal incision in the rightmid-thigh, the right superficial femoral artery was dissected andligated just distally to the origin of the deep femoral artery.

Limb Function and Muscle AtrophyFor a clinical evaluation of the function of the ischemic hind limb theactive movement of the right foot was scored (1�no use; 2�stand-ing; 3�normal use without spreading toes; 4�normal use), aspreviously described.21 For evaluating the extent of atrophy theweights of the excised left and right m. gastrocnemius weredetermined.

Tissue PerfusionRelative hemoglobin oxygen saturation measurements were per-formed using an AbTisSpec spectrometer (LEA, Medizintecknik)placing the probe alternately on the left and right foot. Laser Dopplerperfusion imaging (LDI; Moor Instruments Ltd) was used forrecording serial relative blood flow measurements. The whole regionof the foot was analyzed on either side. All measurements wereperformed in a preheated chamber (37°C) after 5 minutes of warmingand under the influence of a mixture of ketamin (15 mg/kg) andxylazin (82 mg/kg). Measurements are expressed as right-to-leftratios.

Collateral-Dependent Blood FlowBlood flow in the three main arteries of both m. gastrocnemius wasanalyzed by Magnetic Resonance Imaging (MRI), as previouslydescribed.22,23 For further details, see supplemental methods. Asubset of mice from WT and eNOS�/� groups was studied afterintraperitoneal injection of 5 mg/kg of SNAP (S-nitroso-N-acetyl-penicillamine; Sigma).

HistologyFor evaluating capillary density, cryosections (7 �m) were cut fromthe m. gastrocnemius and stained with an antibody against lectin(BS-1; Sigma). Capillaries and muscle fibers were counted and dataexpressed as capillary-to-muscle fiber ratio. We used 2 differenttechniques for collateral artery morphometry. In 1 set of mice, forultrastructure, collateral vessels in the m. adductor were isolated,dissected, and embedded as previously described.7 Ultra-thin sec-tions (1 �m) were cut and stained with toluidine blue. Collateralarteries (ligated side) and preexisting arterioles (non-ligated side)were then measured with NIH software, and subsequently diameters,wall areas, and wall thicknesses were calculated. In another set ofmice, the complete m. adductor was dissected and embedded.Cryosections (7 �m) were cut and immunostained with an antibodyagainst �-SM-actin (Sigma). In both ligated and non-ligated musclesarterioles were identified and measured as above. For further details,see online data supplement.

StatisticsStatistical analysis of all data were performed using 1-way ANOVAfollowed by a multiple comparison test. Data are reported asmeans�SEM. Statistical significance was accepted when P�0.05(2-tailed).

ResultseNOS-Overexpressing Mice

Tissue PerfusionHemoglobin oxygen saturation measurements revealed thatoxygen saturation, a marker for tissue perfusion, was onlysignificantly increased in eNOStg mice immediately afterligation, compared with WT (R/L post-ligation: 0.56�0.06versus 0.24�0.05 in eNOStg and WT, respectively,P�0.001; Figure 1A). Equally, LDI measurements of the feetonly showed a significant increase in relative blood flow ineNOStg mice immediately after ligation, compared with WT

Mees et al Normal Collateral Growth in Absence or Excess of eNOS 1927


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(R/L post-ligation: 0.33�0.04 versus 0.13�0.02, P�0.01;Figure 1B).

Collateral-Dependent Blood FlowThe inflow of blood in the calf arteries is directly dependenton collateral flow in the upper part of the leg and wasmeasured by MRI. In eNOStg mice collateral-dependentblood flow was significantly increased only immediately afterligation, matching tissue perfusion findings in the foot (R/Lpost-ligation: 0.12�0.01 versus 0.05�0.01 in eNOStg andWT, respectively; P�0.001; Figure 1C).

The above measurements, showing differences in bloodflow recovery between eNOStg and WT mice only immedi-ately after the femoral ligation, suggested an increased acute

maximal vasodilation in eNOStg mice but no beneficialeffects of eNOS overexpression on arteriogenesis.

eNOS-Deficient Mice

Limb Function and Muscle AtrophyLimb function, assessed by a foot movement score, recoveredcompletely to normal within 2 weeks in WT mice, whereaslimb function of eNOS�/� mice did not even reach normallevels during complete follow-up (Figure 2A). Similarly, toenecrosis and autoamputation were commonly seen in theeNOS�/� group, in contrary to the mice in the other groups(data not shown). Also, significant more m. gastrocnemiusatrophy was seen in the eNOS�/� mice compared with WTmice (R/L muscle weight: 0.94�0.02 versus 0.78�0.04 inWT and eNOS�/�, respectively; P�0.01; Figure 2B).

Tissue PerfusionOxygen saturation remained significantly impaired ineNOS�/� mice during complete follow-up (Figure 2C). Asexpected, relative blood flow in the feet, assessed by LDI,remained equally impaired until 3 weeks after ligation(Figure 2D).

Collateral-Dependent Blood FlowIn contrast with tissue perfusion findings, a decrease incollateral-dependent blood flow was found only up to 7 daysafter surgery in eNOS�/� mice, when compared with WT,suggesting normal or possibly somewhat delayed collateralartery growth in eNOS�/� mice (R/L blood flow at day 7:1.02�0.07 versus 0.69�0.08 in WT and eNOS�/�, respec-tively; P�0.01; at day 14: 0.81�0.04 versus 0.83�0.08 inWT and eNOS�/�; Figure 3A). In eNOS�/� mice the bloodflow even continued to increase and was significantly higherthan in WT mice at 3 weeks. To circumvent the effects ofvasoconstriction in eNOS�/� mice, we systemically adminis-tered at day 7 the NO-donor SNAP to both WT and eNOS�/�

mice and measured collateral-dependent blood flow. In WTmice, SNAP induced vasodilation and thus an increase ofblood flow in the unligated leg, whereas in the ligated leg noincrease in blood flow could be induced, as vasodilation wasalready at its maximal (or submaximal) level (Figure 3B). IneNOS�/� mice, however, SNAP induced vasodilation andincrease of blood flow to the same extent in both legs. Thissuggested that normal functional collateral arteries had grownin the eNOS�/� mice, but that these were unable to vasodilatesufficiently because of impaired NO production.

Collateral MorphometryFinally, to obtain anatomic data of collateral arteries westudied collateral artery growth in the adductor muscle of the3 groups of mice using 2 different histological analyses.

CryosectionsIn complete cross-sections of the adductor muscle conductingarterioles were identified and analyzed (Figure 4A). Diame-ters of preexisting arterioles in the nonligated adductormuscle did not differ between eNOStg, WT, and eNOS�/�

mice (in �m: 37�2 versus 33�3 versus 34�2 in eNOStg,WT, and eNOS�/�, respectively; Figure 4B). Collateral diam-eters significantly and continuously increased after ligation,

Figure 1. A, Time course of oxygen saturation in the feet(n�10). B, Time course of tissue perfusion in the feet measuredby Laser Doppler Imaging (n�10). C, Time course of collateral-dependent blood flow in the calf muscle measured by MRI(n�10). All measurements are expressed as ligated/non-ligatedratios, ***P�0.001 vs WT.

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as compared with preexisting arterioles, and no differencesbetween collateral diameters from eNOStg, WT, andeNOS�/� mice were found at 1 and 3 weeks (in �m: 62�11versus 76�11 versus 67�15 in eNOStg, WT, and eNOS�/�,respectively). Wall areas of preexisting arterioles of eNOStgmice were significantly smaller than in WT mice (in �m2:557�51 versus 958�107 in eNOStg and WT, respectively;P�0.01; Figure 4C). In all 3 groups wall areas significantlyand continuously enlarged after ligation. At 1 and 3 weeksafter ligation no differences were found between the wallareas of collateral arteries from eNOStg, WT, and eNOS�/�

mice (in �m2: 1098�195 versus 1610�240 versus1598�321 in eNOStg, WT, and eNOS�/�, respectively, at 3weeks after ligation). Analysis of the wall thickness ofcollateral arteries revealed the same significant and con-tinuous enlarging after ligation in all 3 groups of mice(Figure 4D). The wall thickness of collateral arteries ineNOStg mice was smaller than in WT mice, both beforeand after ligation. However, no differences in wall thick-ness were observed between eNOS�/� and WT mice,neither before nor after ligation (in �m: 8.4�0.5 versus7.8�0.5 in WT and eNOS�/�, respectively, before ligationand 11.8�1.3 versus 10.7�0.8, in WT and eNOS�/�, at 3weeks after ligation).

Ultra-Thin SectionsIn ultra-thin sections cut from the predilection area forcollateral artery growth in the adductor muscle, preexisting

arterioles or collateral arteries were identified and analyzed(Figure 5A). We did not find any significant differences indiameters and wall area between preexisting arterioles ineNOStg, WT, and eNOS�/� mice (Figure 5B and 5C). Threeweeks after ligation diameters and wall areas from collateralarteries were significantly enlarged in all 3 groups of mice, ascompared with preexisting arterioles. However, as in cryo-sections, between groups no differences were found in col-lateral artery size. These findings confirmed the above bloodflow data and indicate that collateral artery growth wasnormal in conditions of either deficient or increased eNOSactivity.

Capillary DensityCapillary density measurements in the calf muscle confirmedthe arteriogenesis-specificity of our hind limb model. Inunligated calf muscles of eNOStg and eNOS�/� mice capil-lary density was decreased compared with WT mice (incapillary/fiber ratio: 1.01�0.06 versus 1.23�0.05 versus1.00�0.04, for eNOStg, WT, and eNOS�/�, respectively;P�0.01; Figure 5D). After ligation the capillary/fiber ratio incalf muscles in both eNOStg and WT mice did not change,suggesting the absence of an ischemic stimulus for angiogen-esis or vasculogenesis in these mice. In eNOS�/� mice,however, a significant 1.8-fold increase in capillaries wasseen in the ligated leg, implying the presence of angiogenesisand tissue ischemia.

Figure 2. A, Limb function evaluated by active foot movement score (n�6). B, M. gastrocnemius atrophy (n�8). C, Time course of oxy-gen saturation in the feet (n�6). D, Time course of tissue perfusion in the feet measured by laser Doppler imaging (n�6). All measure-ments are expressed as ligated/non-ligated ratios, **P�0.01 and ***P�0.001 vs WT.



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DiscussionIn the present study we evaluated the role of eNOS in a hindlimb model, specific for arteriogenesis. Firstly, to evaluate thepossible benefits of elevated eNOS activity on collateralgrowth we compared transgenic mice overexpressing humaneNOS with WT control mice. We only found a beneficialeffect of overexpression of eNOS in the acute phase of bloodflow recovery after femoral occlusion. At all later time pointsduring follow-up blood flow recovery was equal in eNOStgand WT mice. Histological analysis revealed no differencesin collateral artery growth between the 2 groups of mice. In apreviously published study performed with a different trans-genic mouse overexpressing (bovine) eNOS beneficial effectsof eNOS overexpression on blood flow recovery after arterialocclusion were found during complete follow-up.24 In anotherstudy adenoviral eNOS-gene transfer in the ischemic hindlimb of rats resulted in improved blood flow recovery andincrease in capillary density.25 However, in both studies amuch more severe hind limb ischemia model was used,causing large ischemia and tissue damage in the lower limband inducing both arteriogenesis in the upper limb and

angiogenesis and vasculogenesis in the lower limb. There-fore, beneficial effects of eNOS overexpression could not beextrapolated to 1 single type of vascular growth. Our findingssuggest that the beneficial effects of eNOS overexpression onblood flow recovery in a severe hind limb ischemia model arenot the result of increased arteriogenesis, but rather increasedangiogenesis or vasculogenesis. Besides, the beneficial effectof eNOS overexpression in our study immediately afterfemoral occlusion suggests an important role for NO-mediated vasodilation in the initial phase of blood flowrecovery, when vascular growth cannot have reached asubstantial level yet.

Previous studies suggesting a positive role for eNOS inarteriogenesis have used different models, either the severemurine hind limb ischemia model8 or an exercise based-model in rats.26 However, in these studies arteriogenesis andangiogenesis were both present and not analyzed separately.The distal femoral artery ligation model used in our study isan in vivo model specific for collateral artery growth. Inearlier studies of our group minimal hypoxia and ischemiawhere found in the lower leg.7,27 Capillary density data fromour study also suggest the absence of an ischemic stimulus inthe lower leg, because no increase of capillary growth wasseen after femoral occlusion in control animals. Therefore,our present study is the only study on eNOS and collateralartery growth using an in vivo model specific for this type ofvascular growth based on acute increase of shear stress afterarterial occlusion.

As we did not find beneficial effects of eNOS overexpres-sion on arteriogenesis, we were interested to evaluate theeffect of absence of eNOS activity on arteriogenesis and thuscompared eNOS�/� and WT mice using the same hind limbmodel and measurements. In earlier studies using eNOS�/�

mice and the severe hind limb ischemia model it was alreadydemonstrated that eNOS�/� mice had a very poor blood flowrecovery, suggested to be caused by impaired angiogenesis orvasculogenesis.9,10 Equally, in our study distal femoral arteryligation caused a significantly reduced blood flow recovery aswell as tissue damage in eNOS�/� mice, despite our observa-tions of an increase in angiogenesis and normal arteriogenesisin eNOS�/� mice compared with WT. We demonstrated thatthe impaired blood flow recovery was not caused by impairedarteriogenesis, but by insufficient vasodilation in the earlyrecovery phase, before collateral arteries were completelyformed.

Using MRI, a relatively new technique which measuresabsolute (and largely deep muscular) blood flow and is lessinfluenced by changes in skin blood flow as LDI, we found ineNOS�/� mice a decreased collateral-dependent blood flowonly in the first week after ligation. In eNOS�/� mice,collateral-dependent blood flow continued to increase during3 weeks of follow up, whereas in WT mice blood flowremained the same or was slightly decreased after 2 weeks.We suspect that after 2 weeks vasodilation is reduced in WTmice as growing collaterals adequately provide bulk flow tothe distal leg, resulting in equal (or slightly decreased) netcollateral blood flow after 3 weeks. In the eNOS�/� mice,however, in the absence of vasodilation collateral blood flowcontinues to increase as collaterals continue to enlarge.

Figure 3. A, Time course of collateral-dependent blood flow inthe calf muscle measured by MRI (n�10). Measurements areexpressed as ligated/non-ligated ratio, *P�0.05, **P�0.01, and***P�0.001 vs WT. B, Collateral-dependent blood flow beforeand after administration of NO-donor SNAP 7 days after femoralartery ligation (n�13). Measurements are expressed as arbitraryunits, #P�0.05 and ##P�0.01 vs before SNAP, ***P�0.001 vs WT.



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Consequently, administration of the NO-donor SNAP con-firmed the insufficient vasodilation of collateral arteries ineNOS�/� mice caused by the absence of eNOS activity.

To obtain conclusive anatomic data of collateral arterieswe extensively studied collateral artery growth in the adduc-tor muscle after maximal vasodilation using 2 differenthistological analyses. Cryosections were immunohistochemi-cally stained and used to study collateral artery growth in the

general overview of the whole adductor muscle. For ultra-thinsections only the predilection area for collateral artery growthwas isolated. This isolation procedure and the thinness ofthese sections permitted us to analyze a smaller number ofvessels in more detail. Both histological analyses revealednormal collateral artery growth in eNOS�/� mice after 1 and3 weeks, suggesting a crucial role for vascular tonus in theearly phase after femoral ligation. Finally, the tissue damage

Figure 4. A, Representative photographs of cryosections with a collateral artery (A) in the adductor muscle of eNOStg, wild-type, andeNOS�/� mice at different time points. Note the accompanying vein (V). Red�artery wall stained with anti–�-SMC actin, blue�nucleistained with Dapi (magnification 400�). B, C, and D, Morphometric measurements of diameter, wall area, and wall thickness of collat-eral arteries and preexisting arterioles (n�5). #P�0.05, ##P�0.01, and ###P�0.001 vs preexistent, *P�0.05, **P�0.01, and ***P�0.001 vsWT.



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in the lower limbs of eNOS�/� mice appeared irreversible,because at 3 weeks of follow up muscle weights weredecreased, tissue perfusion of the feet remained impaired, andcollateral-dependent blood flow was still increasing. Thisirreversibility stresses the essential role of NO-mediatedvasodilation.

Our findings confirm that eNOS activity is essential for aneffective restoration of blood flow after femoral artery occlu-sion, however not by stimulation of arteriogenesis but byinducing NO-dependent vasodilation of the collateral-dependent peripheral vessels. Because arteriogenesis itself isnot an immediate process and thus cannot protect tissue fromischemic injury in the acute phase, the combination of bothadequate vasodilation (of peripheral collateral vessels) in theacute phase and vascular (ie, collateral artery) growth for

continuation is necessary for successful blood flow recoveryand tissue salvage.

Clinical trials aiming for therapeutic neovascularization bystimulating vascular growth using different growth factorshave shown disappointing results.5,6 These disappointingresults might be attributable to the preexistent endothelialdysfunction in patients with atherosclerosis or diabetes.Endothelial dysfunction causes decreased vasoreactivity as aresult of a reduced bioavailability of NO. Therefore, theremight be (partial) analogies between these patients andeNOS�/� mice, in which arteriogenesis is not impaired but theinability to sufficiently vasodilate resulted in severe ischemicdamage. However, similar to the approach to stimulatevascular growth only, stimulation of vasodilation only mightnot be sufficient to adequately restore blood flow distally

Figure 5. A, Representative photographs of ultra-thin sections with a collateral artery (A) in the adductor muscle of eNOStg, wild-type,and eNOS�/� mice, 21 days after femoral artery ligation (magnification 630�). Note the accompanying vein (V) or nerve (N). In all 3 col-laterals at least 1 smooth-muscle cell nucleus (*) can be identified. B and C, Morphometric measurements of diameter and wall area ofcollateral arteries and preexisting arterioles (n�5). #P�0.05, ##P�0.01, and ###P�0.001 vs preexistent. D, Capillary density measure-ments in the calf muscle (n�5). Measurements are expressed as capillary/fiber ratio, #P�0.05 and ###P�0.001 vs preexistent, *P�0.05and **P�0.01 vs WT.



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from the occlusion in a clinical situation of chronic ischemiain the lower limb. A combination of stimulation of vasculargrowth and vasodilation could be essential for successfultherapeutic neovascularization, specifically in conditionswhere endothelial dysfunction is present (ie, hypercholester-olemia and diabetes). We are currently investigating thishypothesis using both diabetic and hypercholesterolemicmice.

In summary, eNOS overexpression did not have beneficialeffects for collateral artery growth. eNOS deficiency causedsevere impaired blood flow recovery after arterial occlusioncaused by insufficient vasodilation of collateral-dependentvessels, whereas collateral artery growth was intact. There-fore, eNOS activity is essential for an effective restoration ofblood flow after femoral artery occlusion, however not bystimulation of arteriogenesis but by inducing NO-dependentvasodilation of the collateral-dependent peripheral vessels.

Sources of FundingThis study was financially supported by The Netherlands Organiza-tion for Health Research and Development (AGIKO stipend 920-0-291 to B.M.), by the Erasmus MC Rotterdam (“Breedtestrategie”),and by the Foundation “De Drie Lichten” in the Netherlands.

DisclosuresNone.

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18. Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, Epstein SE.Marrow-derived stromal cells express genes encoding a broad spectrumof arteriogenic cytokines and promote in vitro and in vivo arteriogenesisthrough paracrine mechanisms. Circ Res. 2004;94:678–685.

19. van Haperen R, de Waard M, van Deel E, Mees B, Kutryk M, van AkenT, Hamming J, Grosveld F, Duncker DJ, de Crom R. Reduction of bloodpressure, plasma cholesterol, and atherosclerosis by elevated endothelialnitric oxide. J Biol Chem. 2002;277:48803–48807.

20. van Haperen R, Cheng C, Mees BM, van Deel E, de Waard M, vanDamme LC, van Gent T, van Aken T, Krams R, Duncker DJ, de Crom R.Functional expression of endothelial nitric oxide synthase fused to greenfluorescent protein in transgenic mice. Am J Pathol. 2003;163:1677–1686.

21. Heil M, Ziegelhoeffer T, Wagner S, Fernandez B, Helisch A, Martin S,Tribulova S, Kuziel WA, Bachmann G, Schaper W. Collateral arterygrowth (arteriogenesis) after experimental arterial occlusion is impairedin mice lacking CC-chemokine receptor-2. Circ Res. 2004;94:671–677.

22. Wagner S, Helisch A, Ziegelhoeffer T, Bachmann G, Schaper W.Magnetic resonance angiography of collateral vessels in a murine femoralartery ligation model. NMR Biomed. 2004;17:21–27.

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24. Amano K, Matsubara H, Iba O, Okigaki M, Fujiyama S, Imada T, KojimaH, Nozawa Y, Kawashima S, Yokoyama M, Iwasaka T. Enhancement ofischemia-induced angiogenesis by eNOS overexpression. Hypertension.2003;41:156–162.

25. Smith RS, Jr., Lin KF, Agata J, Chao L, Chao J. Human endothelial nitricoxide synthase gene delivery promotes angiogenesis in a rat model ofhind limb ischemia. Arterioscler Thromb Vasc Biol. 2002;22:1279–1285.

26. Lloyd PG, Yang HT, Terjung RL. Arteriogenesis and angiogenesis in ratischemic hind limb: role of nitric oxide. Am J Physiol Heart Circ Physiol.2001;281:H2528–H2538.

27. Helisch A, Wagner S, Khan N, Drinane M, Wolfram S, Heil M,Ziegelhoeffer T, Brandt U, Pearlman JD, Swartz HM, Schaper W. Impactof mouse strain differences in innate hind limb collateral vasculature.Arterioscler Thromb Vasc Biol. 2006;26:520–526.



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Sawa Kostin, Matthias Heil, Rini de Crom and Wolfgang SchaperBarend Mees, Shawn Wagner, Elena Ninci, Silvia Tribulova, Sandra Martin, Rien van Haperen,

Flow Recovery but not for ArteriogenesisEndothelial Nitric Oxide Synthase Activity Is Essential for Vasodilation During Blood

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2007 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.107.1453752007;27:1926-1933; originally published online June 7, 2007;Arterioscler Thromb Vasc Biol.

http://atvb.ahajournals.org/content/27/9/1926World Wide Web at:

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/content/33/3/e102.full.pdfAn erratum has been published regarding this article. Please see the attached page for:

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document. Question and AnswerPermissions and Rightspage under Services. Further information about this process is available in the

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e102

Correction

(Arterioscler Thromb Vasc Biol. 2013;33:e102.)© 2013 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATV.0b013e318287c067

In the article by Mees et al, which appeared in the September 2007 issue of the journal (Arterioscler Thromb Vasc Biol. 2007;27:1926–1933. DOI: 10.1161/ATVBAHA.107.145375), there was an error in Figure 2. The correct version of the figure appears below. The online version of the article has been corrected.

Uma

Online Methods Data Supplement

Transgenic mice overexpressing eNOS-GFP1

A genomic DNA fragment including 6 kb of 5’ sequence, the complete eNOS

gene including its native promotor, and 3 kb of 3’ sequence was isolated from a

human genomic cosmid library. At the STOP codon of the eNOS gene, a linker

was introduced that allowed the in-frame insertion of a BamHI-NotI DNA

fragment encoding eGFP which was derived from the pEGFP-N1 plasmid (BD

Biosciences Clontech). A solution of 1–2 µg/ml DNA was used for microinjection

of fertilized oocytes from FVB donor mice and transplanted into the oviducts of

pseudopregnant B10 x CBA mice. Founder mice and offspring were genotyped

by PCR. Mice were backcrossed to C57Bl6 for at least ten generations (>99%

C57Bl/6). Transgenic mice used in the present study were hemizygous.

Collateral-dependent blood flow

A 7.05T Bruker PharmaScan MR small animal scanner was used for imaging

blood flow in the m. gastrocnemius, as previously described2-5. A fast gradient

echo sequence with a slice thickness of 0.62 mm and a repetition time of 20 ms

was used to obtain six axial slices along the length of the muscle with an in-plane

resolution of 100 x 100 µm for a 2.56 x 2.56 cm imaging area with 16 averages

resulting in a total imaging time of 5 minutes. A 90-degree flip angle was used to

obtain signal from primarily just the blood flow in the larger vessels. For the

I

parameters used, the signal intensity obtained is linearly proportional to the blood

flow.

A specially designed mouse imaging quadrature probe was used to achieve an

adequate signal-to-noise ratio for all experiments. The probe contained a water

jacket to allow for mouse heating while in the animal scanner. The ambient

temperature was set to 37°C. All mice were preheated for at least five minutes at

37°C before any imaging was done. Right-to-left blood flow ratios were

calculated from the intensities obtained from the corresponding right and left m.

gastrocnemius. The six slices were averaged to obtain an average blood flow

ratio for each mouse at each time point.

Perfusion Fixation and Tissue Sampling

At day 7 and 21 after surgery, mice were euthanized. After administration of

ketamin/xylazin and heparin (625 IU), we cannulated the left ventricle and

perfused the mouse under constant pressure of 100 mm Hg during ten minutes

with PBS buffer, containing 0.5% albumin and 0.1% adenosin to achieve

maximal vasodilation. Subsequently, we perfused with the fixative, 2%

paraformaldehyde, for another ten minutes. For cryo-sections, the complete m.

adductor and m. gastrocnemius of both legs were excised. The tissue was post-

fixed in 2% paraformaldehyde and cryo-protected overnight in a 10% sucrose

solution at 4°C. After 24 hours the tissue was embedded in Tissue-Tek (Sakura

Finetek) and snap-frozen in methylbutane chilled with liquid nitrogen. For ultra-

thin sections, the predilection place for the main collateral arteries in the adductor

II

muscle was identified and a superficial rectangle containing these collaterals

(right leg) or the pre-existing arterioles (left leg) was excised, as previously

described6. The tissue was post-fixed in 3% glutaraldehyde, then in 1% OsO4,

dehydrated in alcohols and embedded in epon using the Lynx Microscope Tissue

Processor (Reichert).

Capillary Density

Using the Leica CM 3000 Cryo-microtome cryo-sections (7 µm) were cut from the

upper-, middle- and lower part of the m. gastrocnemius. For identifying capillaries

and not interfering with the GFP signal in the eNOS-GFP mice we stained

sections with a monoclonal TRITC-conjugated antibody against lectin (BS-1,

Sigma). The immunoreactions were visualized with a Leica DMLD fluorescence

microscope and photographed with the accompanying digital camera. In three

fields per section and three sections per part of the muscle the amount of

capillaries and muscle fibers, identified by auto-fluorescence, was counted using

the NIH software. The mean capillary-to-muscle fiber ratio was calculated per

muscle.

Collateral Artery Morphometry Ultra-thin sections

Ultra-thin sections were cut from all four parts of the excised rectangle of

adductor muscle with the Ultracut Microtome (Reichert) to a thickness of 1 µm.

Sections were stained with toluidine blue and analyzed with the Leica DMLD

microscope. Criteria for identifying a collateral artery were characteristic location,

III

proliferating endothelial and smooth-muscle cells, surrounding vein and/or nerve,

continuous internal lamina elastica and size. Using the NIH image software we

measured the internal and external perimeter of the intima/media, which enabled

us to calculate diameter, wall area and wall thickness.

Cryo-sections

Serial cryo-sections (7 µm) were cut and immediately stained with toluidine blue

for identifying the first level of insertion of collateral arteries into the superficial

femoral artery. From this level on consecutive sections were cut, mounted and

immunostained with a monoclonal CY3-conjugated antibody against α-SM-actin

(Sigma). Collateral arteries (ligated side) and pre-existing arteries (non-ligated

side) were identified by the presence of a pronounced α-SM-actin positive media.

Internal and external perimeters of intima/media were measured as in semi-thin

sections and subsequently diameter and wall area were calculated.

IV

References

1. van Haperen R, Cheng C, Mees BM, van Deel E, de Waard M, van

Damme LC, van Gent T, van Aken T, Krams R, Duncker DJ, de Crom R.

Functional expression of endothelial nitric oxide synthase fused to green

fluorescent protein in transgenic mice. Am J Pathol. 2003;163:1677-1686.

2. Heil M, Ziegelhoeffer T, Wagner S, Fernandez B, Helisch A, Martin S,

Tribulova S, Kuziel WA, Bachmann G, Schaper W. Collateral artery

growth (arteriogenesis) after experimental arterial occlusion is impaired in

mice lacking CC-chemokine receptor-2. Circ Res. 2004;94:671-677.

3. Wagner S, Helisch A, Ziegelhoeffer T, Bachmann G, Schaper W.

Magnetic resonance angiography of collateral vessels in a murine femoral

artery ligation model. NMR Biomed. 2004;17:21-27.

4. Wagner S, Helisch A, Bachmann G, Schaper W. Time-of-flight quantitative

measurements of blood flow in mouse hindlimbs. J Magn Reson Imaging.

2004;19:468-474.

5. Helisch A, Wagner S, Khan N, Drinane M, Wolfram S, Heil M,

Ziegelhoeffer T, Brandt U, Pearlman JD, Swartz HM, Schaper W. Impact

of mouse strain differences in innate hindlimb collateral vasculature.

Arterioscler Thromb Vasc Biol. 2006;26:520-526.

6. Scholz D, Ziegelhoeffer T, Helisch A, Wagner S, Friedrich C, Podzuweit T,

Schaper W. Contribution of arteriogenesis and angiogenesis to

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endothelial nitric oxide synthase activity is essential...

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