Nitric Oxide Deficit Drives Intimal Hyperplasia in Mouse ...

Eur J Vasc Endovasc Surg (2016) -, 1e10

Nitric Oxide Deficit Drives Intimal Hyperplasia in Mouse Models ofHypertension, 5

F. Allagnat a, J.-A. Haefliger a, M. Lambelet a, A. Longchamp a,b,c, X. Bérard d, L. Mazzolai e, J.-M. Corpataux a,f, S. Déglise a,*,f

a Department of Vascular Surgery, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerlandb Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USAc Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, USAd Department of Vascular Surgery, Hôpital Pellegrin, Bordeaux, Francee Department of Angiology, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland

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WHAT THIS PAPER ADDS
The long-term patency of revascularization surgery in patients with peripheral artery disease is plagued byrestenosis caused by intimal hyperplasia (IH), leading to progressive vessel re-occlusion. Although correlationsexist between hypertension and IH, rigorous evidence and mechanistic insights are lacking. To elucidate themolecular mechanisms by which hypertension aggravates IH, IH formation after complete ligation of the carotidartery was compared in three different mouse models of hypertension. These results provide evidence that NOdeficiency is the main driving force accelerating the development of IH in the context of hypertension.
Objective: To evaluate the impact of different types of hypertension on the development of intimal hyperplasia(IH).Method: Genetic, surgical, and pharmacological models of hypertension were used to compare IH formation in amurine model of carotid artery ligation (CAL). CAL was performed in normotensive WT male mice and in threemouse models of hypertension: (1) L-NAME (Nu-nitro-l-arginine-methyl-ester) treatment for 2 weeks prior toCAL to instate renin-independent hypertension; (2) 2K1C (two kidneys, one clip) surgery 1 week prior to CAL toinduce renin-dependent hypertension; (3) Cx40�/� mice, a genetic model of renin-dependent hypertension.Mice were sacrificed prior to CAL or 3, 14, or 28 days post CAL. Data collection included tail blood pressuremeasurements, and morphometric and histological assessment of the ligated carotids.Results: CAL triggered the formation of a VSMC-rich neointima layer after 14e28 days, which was increased in allhypertensive mice. Despite similarly increased blood pressure, L-NAME treated mice displayed more IH than allother hypertensive groups. In addition, L-NAME induced hypertension triggered more cell proliferation andrecruitment of CD45 positive inflammatory cells to the ligated vessel wall compared with Cx40�/� ornormotensive WT mice.Conclusions: NO deficiency is a major aspect of vascular inflammation, VSMC proliferation, and IH in hypertensiveconditions.� 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.Article history: Received 9 November 2015, Accepted 29 January 2016, Available online XXXKeywords: Intimal hyperplasia, Hypertension, Proliferation, Inflammation, Carotid artery ligation, NO

INTRODUCTION

Prevalence of peripheral artery disease (PAD) continues torise worldwide, largely caused by a combination of aging,

is study was presented as an oral communication at the Springg of the European Society for Vascular Surgery held in Frankfurt, 2915.. Corpataux and S. Déglise contributed equally to this work.rresponding author. CHUV, BH15e303, Bugnon 21, 1011 Lausanne,land.il address: [email protected] (S. Déglise).-5884/� 2016 European Society for Vascular Surgery. Published byr Ltd. All rights reserved.//dx.doi.org/10.1016/j.ejvs.2016.01.024

cite this article in press as: Allagnat F, et al., Nitric Oxide Deficit Drives Ilar and Endovascular Surgery (2016), http://dx.doi.org/10.1016/j.ejvs.20

smoking, hypertension, and diabetes mellitus.1,2 Contem-porary surgical treatments for PAD include endovascularand open approaches. However, despite remarkable tech-nological advances, along with better peri- and post-operative management, intimal hyperplasia (IH) leading torestenosis remains a major threat to the success of theseprocedures.3 IH is an exaggerated healing response to thevessel wall injury resulting in inflammation, VSMC de-differentiation, migration, and proliferation leading to theformation of a neointima layer.4 Hypertension is a majorindependent risk factor for the development of vascularocclusive disease, stroke, and in-stent coronary reste-nosis,1,2,5e8 but the molecular mechanisms linking hyper-tension to IH formation remain largely unknown.

ntimal Hyperplasia in Mouse Models of Hypertension, European Journal of16.01.024

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http://dx.doi.org/10.1016/j.ejvs.2016.01.024

2 F. Allagnat et al.

Blood pressure is primarily controlled by the renalendocrine renineangiotensinealdosterone (RAA) system,which increases systemic vasoconstriction and blood pres-sure via angiotensin II (AngII), as a response to reduction inrenal perfusion. Alternatively, the endothelial layer regu-lates vascular tone through the release of vasodilatingmolecules such as nitric oxide (NO).9 Besides their acuteeffects on vessel tone, AngII and NO also induce long-termchanges in vessel structure. AngII stimulates molecularmechanisms that promote IH,10,11 whereas NO acts atvarious levels to limit IH development.12 To better under-stand the mechanisms leading to IH in hypertensive con-ditions independently of pressure, development of IH usingthe murine model of carotid artery ligation13 was comparedin three different mouse models of hypertension. The re-sults provide evidence that although AngII facilitates IHdevelopment, NO deficiency is the main driver of IH.

METHOD

Experimental design

In this study the impact of renin-dependent hypertensionand NO-dependent hypertension on IH was compared usingthe murine model of carotid artery ligation (CAL) developedby Kumar et al.13 IH development was monitored in controlmice and three mouse models of hypertension. NO-dependent hypertension was induced by treatment withthe inhibitor of NO production Nu-nitro-l-arginine methylester (L-NAME). Two models of AngII-dependent hyperten-sion were also studied: the Cx40 deficient mice (Cx40�/�),which constitutes a genetic model of AngII-dependent hy-pertension through constitutive secretion of renin,14,15 andthe surgical two kidneys, one clip (2K1C) model of hyper-tension, in which the unilateral stenosis of one renal arteryleads to kidney hypoperfusion and increased levels ofcirculating renin.15,16

Animal models

All experiments were performed using male mice of C57BL/6J genetic background. Cx40�/� mice (a gift from Dr. K.Willecke17) were bred and genotyped as previouslydescribed.15,16 The L-NAME model was achieved as previ-ously described16 by adding Nu-Nitro-l-arginine methylester hydrochloride (SigmaeAldrich Chemie GmbH, Buchs,Switzerland) to drinking water at 80 mg/kg/day during 2weeks before surgery and until sacrifice. The two-kidney,one clip (2K1C) model was performed as previously pub-lished.15,16 Briefly, the left renal artery was clipped with a0.12 mm internal diameter U-shaped silver clip 1 week priorto CAL surgery.

The carotid artery ligation (CAL) was performed accordingto the protocol developed by Kumar et al.13 Briefly, 2 monthold male mice were anesthetized with ketamin (100 mg/kg)(Ketasol-100, Gräub E. Dr. AG, Bern, Switzerland) and xylazin(15 mg/kg) (Rompun, Provet AG, Lyssach, Switzerland). Theleft common carotid artery was dissected through a smallcervicotomy and ligated with Prolene 7.0 (Johnson &

Please cite this article in press as: Allagnat F, et al., Nitric Oxide Deficit Drives InVascular and Endovascular Surgery (2016), http://dx.doi.org/10.1016/j.ejvs.20

Johnson AG, Ethicon, Zug, Switzerland) just below the ca-rotid bifurcation. Buprenorphine (0.05 mg/kg Temgesic,Reckitt Benckiser AG, Switzerland) was provided as a post-operative analgesic. Mice were sacrificed by cervical dislo-cation, perfused with PBS followed by buffered formol 4%through the left ventricle prior to CAL (D0) for reference or3 (D3), 14 (D14), or 28 (D28) days after CAL. Hearts wereweighed and the cardiac weight index (heart weight in mgover total body weight in g) calculated as previouslydescribed.16,18

Measurement of blood pressure

Systolic blood pressure was monitored in 4 day trained miceby a non-invasive computerized tail cuff method (BP-2000,Visitech Systems Inc.). The system automatically performscuff inflation and deflation cycles and records pulse rate andblood pressure. Data are average of systolic blood pressureover a 4 day period as previously described.16,18

Histomorphometry

Both ligated left and right contralateral carotids werecarefully dissected and paraffin embedded. Six mm sectionsof the ligated carotid artery were cut from the ligature to-wards the aortic arch and stained with hematoxylin eosin(HE), and van Gieson elastic laminae (VGEL) for histologicand morphometric analysis. A standardized reference pointwas set in the portion of the carotid where ligature did notdistort the vessel wall and where the elastic lamina wasintact. This point was situated between 0.05 and 0.15 mmdistal to the ligature. Cross sections at every 300 mm and upto 2 mm from the reference point were morphometricallyanalyzed using the QWin software (Leica Microsystems).Total vessel area, lumen circumferences, internal elasticlamina (IEL), and external elastic lamina (EEL) thickness andarea were measured by tracing along the luminal surface,IEL, and EEL on at least six cross sections every 300 mm. Forintimal and medial thickness or area, 72 (12 measures/crosssection on six cross sections) measurements were per-formed. Intimal area was calculated by subtracting theluminal area from the IEL area. Neointimal thickness wasdefined as the distance between the lumen and the IEL. Themedia thickness was defined as the distance between theIEL and the EEL. All measures were performed by twoblinded, independent investigators. Similar methodologyand measurements were performed on control shamoperated animals (D0) and contralateral carotids.

Immunohistochemistry

Carotid paraffin sections were immunostained using CD45(clone 30-F11, 550539, Pharmingen Deutschland GmbH),alpha smooth muscle actin (a-SMA; Abcam, ab5697,Lucerna chem. AG, Luzern, Switzerland), alpha skeletal actin(a-SKA; Novus Biologicals, NB100e91648, Cambridge, UK),proliferating cell nuclear antigen (PCNA; M087901, Dako,Baar, Switzerland), followed by the appropriate avidin-biotinylated horseradish peroxidase complex (VectastainElite ABC kit, USA), and counterstained with hemalun. The

timal Hyperplasia in Mouse Models of Hypertension, European Journal of16.01.024

Nitric oxide and Intimal Hyperplasia 3

PCNA- or CD45-positive signals were evaluated automati-cally using the ImageJ software on 10 slides per carotid andthree to five carotids per condition.19,20 PCNA or CD45 DAB(3,30-diaminobenzidine)-positive signals were quantified asfollows: images were converted into 16 bit and a colorthresholding based on red hue was applied to select onlythe brown staining. The selected staining was processedinto a binary image and the number of positive pixelsdetermined. This signal was normalized to the total pixelnumber composing the carotid section and expressed as apositive pixels/total vein pixels ratio.20

Statistical analysis

All experiments were quantitatively analyzed using Graph-Pad Prism 6, and results are shown as mean � standarderror of the mean (SEM), or box plots � minemax intervals.

Table 1. Systolic blood pressure and cardiac weight index innormotensive and hypertensive mice.

Mouse model Systolic pressure(mmHg)

Cardiac weightindex (mg/g)

Normotensive 96.1 � 2.69 5.4 � 0.1L-NAME 110.2 � 5.5a 6.4 � 0.2a

2K1C 116.0 � 3.2a 6.6 � 0.3a

Cx40�/� 126.1 � 3.0a 6.8 � 0.2a

a p < .001 versus normotensive mice.

Figure 1. Hypertension and artery ligation stimulate hyperplasia and hGieson elastic lamina (VGEL) staining of non-ligated (D0) or 28 days (D2mice, Cx40�/� mice or 2K1C-operated mice. L ¼ lumen; M ¼ media.thickness are presented as a box plot of 25e75 percentile � minema��p < .001 versus respective D0 group. #p < .05, versus respective N

Please cite this article in press as: Allagnat F, et al., Nitric Oxide Deficit Drives IVascular and Endovascular Surgery (2016), http://dx.doi.org/10.1016/j.ejvs.20

One or two way analysis of variance (ANOVA) were per-formed followed by multiple comparisons using post-hocTukey test.

Ethics statement

All animal care, surgery, and euthanasia procedures wereapproved by the Centre Hospitalier Universitaire Vaudois(CHUV) and the Cantonal Veterinary Office (Service de laConsommation et des Affaires Vétérinaires SCAV-EXPANIM,authorization numbers 2290 and 2832).

RESULTS

Vascular remodeling in ligated carotid arteries innormotensive and hypertensive mice

Different types of hypertension similarly stimulate mediahypertrophy. All hypertensive mice featured similar in-creases in blood pressure and heart weight indexescompared with normotensive (NT) WT mice (Table 1). Aspreviously shown in mice aortas,16 all hypertensive micedisplayed a 20% increase in carotid media thickness (Fig. 1and Supplementary Fig. S1). Media thickness doubled in allmice 14 days after the ligation procedure and remainedconstant thereafter (14e28 days) (Fig. 1). Left CAL had noimpact on the media thickness of the contralateral rightcarotid (Supplementary Fig. S1).

ypertrophy of the media layer of carotids. (A) Representative van8) post carotid ligation in normotensive mice (NT), L-NAME treatedBar represents 20 mm. (B) Morphometric measurements of mediax in six to nine animals per group. ***p < .001 versus NT at D0.T at D14 or D28.



Different types of hypertension differentially stimulateCAL-induced IH formation. Neointimal thickness wasmeasured between 0.5 and 2 mm from the ligature 14 and28 days after CAL in normotensive (NT) and hypertensiveanimals (Supplementary Fig. S2). Areas under the curve ofneointima thickness were then calculated (Fig. 2). As ex-pected, in NTmice,13 CAL induced a linear development of aneointima layer 14e28 days post ligation. Carotids from

Figure 2. Hypertension increases intimal hyperplasia. (A) Representativedays (D28) post carotid ligation in normotensive mice (NT), L-NAME-treaL ¼ lumen; M ¼ media. Bar represents 100 mm. (B) Morphometric mea(AUC) of neo-intima area along 0.2 mm of the ligated carotid length. **both Cx40�/� and 2K1C at D14 or D28.


hypertensive mice displayed increased IH, both 14 and 28days post ligation. Neointimal development increasedmildly in Cx40�/� and 2K1C mice, whereas L-NAMEtreated mice had accelerated and more substantial IHdevelopment (Fig. 2).

In NT mice, CAL triggered a 15e20% reduction of theouter vessel circumference (EEL) after 14 or 28 days,respectively. Together with neointima formation (Fig. 3A

VGEL staining of non-ligated (D0) or ligated carotids 14 (D14) or 28ted mice, Cx40�/� mice, or 2K1C-operated mice. NI ¼ neointima;surements of intima thickness. Data represent area under the curvep < .01 versus respective NT group at D14 or D28. �p < .05 versus


Figure 3. Morphometric parameters are altered in the carotids of hypertensive mice. (A) Representative HE staining of non-ligated (D0), or14 (D14) or 28 days (D28) post carotid ligation in normotensive (NT), L-NAME-treated, Cx40�/�, or 2K1C-operated mice. NI ¼ neointima;L ¼ lumen; M ¼ media. Bar represents 100 mm. (B,C) Morphometric measurements of EEL perimeter (B) and lumen area (C) of non-ligated(D0) or 14 (D14) or 28 days (D28) post carotid ligation. Data represent box plot of 25e75 percentile � minemax of four to nine ex-periments. *p < .05, **p < .01 versus NT at D0; �p < .05, ��p < .001 versus respective D0 group.


and B), reduction in vessel caliber resulted in a 50e70%drop in lumen area in NT mice after 14 or 28 days,respectively (Fig. 3C). Before CAL, the EEL perimeter andlumen area were reduced by 30% in the L-NAME and2K1C models, but not in the Cx40�/� model, whichshowed similar EEL perimeters and lumen areas as NTmice (Fig. 3B and C). As expected the left CAL induced a15e20% drop in EEL perimeter in NT mice after 14 or 28days, respectively, and had no impact on the right carotidEEL perimeter (Supplementary Fig. S2). In contrast, 2K1Cand L-NAME animals presented a 30% increase in EELperimeter following CAL while the EEL perimeter ofCx40�/� mice remained unchanged on CAL (Fig. 3B).Interestingly, the left CAL similarly increased the EEL pe-rimeters of the contralateral carotids in the 2K1C and L-NAME animals compared with NT mice, while the EELperimeter of Cx40�/� mice remained unchanged(Supplementary Fig. S2). Despite the outward remodelingobserved following CAL in L-NAME or 2K1C models, allhypertensive models displayed reduced lumen area of theligated carotid 28 days after CAL because of increasedneointimal thickness (Figs. 2 and 3C). At day 28, thelumen area of ligated carotids was not significantlydifferent between normotensive and hypertensive mice(Fig. 3C).


Angiotensin II-dependent hypertension and CAL areassociated with a switch to proliferative VSMC phenotype

Carotid sections were immunostained with the VSMCmarker a-SMA and the proliferative synthetic VSMC markera-SKA in control condition (D0), and 28 days (D28) afterCAL. Cells composing the media and neointima layers of allmice are largely a-SMA positive, indicating that the neo-intima layer was mainly composed of VSMC-derived cells(Fig. 4A). Both AngII-dependent models of hypertension(Cx40�/� and 2K1C) triggered similar increases in a-SKApositive cells at D0 (Fig. 4B and C), whereas L-NAMEtreatment had no significant effect on a-SKA stainingcompared with NT mice. Following CAL, the proportion ofsynthetic a-SKA positive cells increased in the media andneointima layers of all animal groups. The a-SKA stainingwas significantly greater in all models of hypertension thanin NT animals (Fig. 4B and C).

To further characterize VSMC proliferation, PCNA stainingwas performed at the peak of neointima development, 14days after CAL, in the different models. Quantitativeassessment of the PCNA staining showed a twofold increasein the Cx40�/� and 2K1C mice, and a threefold increase inthe carotid of L-NAME treated mice, compared with NTmice (Fig. 5).


Figure 4. Renin dependent hypertension triggers a switch in VSMC phenotype from contractile to synthetic. Representative alpha smoothmuscle actin (a-SMA; A) or alpha skeletal actin (a-SKA; B) immunostaining of non-ligated (D0) or 28 days (D28) post carotid ligation innormotensive (NT), L-NAME-treated, Cx40�/�, or 2K1C-operated mice. NI ¼ neointima; A ¼ adventitia; L ¼ lumen; M ¼ media. Barrepresents 100 mm. Data are representative of four to nine animals. (C) Quantitative assessment of a-SKA positive area over total carotidarea. Data represent mean� SEM of five to six experiments. **p< .01, ***p< .001 versus control animal (D0); #p< .05, ##p< .01 versusNT at D0; �p < .05, ��p < .01 versus NT at D28.


Lack of nitric oxide promotes vessel wall inflammationfollowing CAL

CAL triggers transient local inflammation 3 and 7 days afterthe procedure.13 To further characterize whether hyper-tension modulates the inflammatory process, CD45 stain-ing was performed 3 days post ligation. NT mice andCx40�/� mice presented similar amounts of CD45 posi-tive cells on the endothelial surface and in the adventitialayer of the ligated carotid (Fig. 6). In contrast, the amountof CD45 positive staining was greatly increased in L-NAMEtreated mice, both on the luminal surface of the vesseland in the adventitia, and CD45 positive cells were alsofound infiltrating the media layer of the ligated carotid(Fig. 6).


DISCUSSION

Using various mouse models of hypertension this studydemonstrates that although increased blood pressure drivesincreased IH, different types of hypertension differentiallyaccelerate IH formation following acute carotid injury. NOdeficiency appears to be critical in this pathological adap-tation, more so than hemodynamic perturbations.

Hypertension drives up various tensions applied to thevessel wall, including shear stress and circumferentialstretch, which trigger both inward and outward vessel wallremodeling.21,22 As expected all models of hypertensionstimulated an initial thickening of the media layer. How-ever, hypertension marginally increased media thicknesscompared with CAL, which nearly doubled media thickness


Figure 5. Hypertension promotes VSMC proliferation in the ligated carotid. (A) Representative PCNA immunostaining of 14 days (D14) postcarotid ligation in normotensive (NT) mice, L-NAME-treated mice, Cx40�/� mice, or 2K1C-operated mice. NI ¼ neointima; L ¼ lumen;M ¼ media. Bar represents 100 mm. (B) Quantitative assessment of PCNA positive area over total carotid area. Data representmean � SEM of four to six animals per group. *p < .05, **p < .01, ***p < .001 versus NT. �p < .05, ��p < .01 versus L-NAME treatedmice.

Figure 6. CD45 positive cells are increased in the ligated carotid of L-NAME treated mice. Representative CD45 immunostainingof ligated carotids 3 days post carotid ligation in normotensive (NT) mice, L-NAME-treated mice, or Cx40�/� mice. Bar represents100 mm. Data are representative of four to nine animals.Bottom right panel display quantitative assessment of CD45 positive area overtotal carotid area in control non-ligated vs. ligated carotid 3 days post carotid ligation. Data represent mean � SEM of four animals pergroup. **p < .05, vs. NT.


Please cite this article in press as: Allagnat F, et al., Nitric Oxide Deficit Drives Intimal Hyperplasia in Mouse Models of Hypertension, European Journal ofVascular and Endovascular Surgery (2016), http://dx.doi.org/10.1016/j.ejvs.2016.01.024


in all models. CAL also greatly reduced vessel diameter innormotensive animals, which is thought to happen as avasoactive response to changes in acute blood flow.13

Although increased blood pressure per se impacted thecarotid wall remodeling, several molecular adaptationswere specific to the type of hypertension and seemed in-dependent of blood pressure. Thus, renin-dependent hy-pertension models (Cx40�/� and 2K1C) featured a-SKApositive cells in their media prior to CAL, suggesting anAngII driven switch from contractile to synthetic prolifer-ative VSMC.23 This is in agreement with previous studiesshowing that AngII specifically promotes VSMC phenotypicswitch, proliferation, and migration, independently ofchanges in hemodynamic forces associated with hyper-tension.23e25 Nevertheless, VSMC proliferation and neo-intima formation were more elevated in the ligated carotidof L-NAME treated mice than in both renin-dependentmodels of hypertension, suggesting that lack of NO, evenin the presence of low AngII circulating levels,26 is sufficientto drive VSMC proliferation. The trauma resulting from theligation in the CAL model is known to trigger an initialtransitory inflammatory response.13 Here it was observedthat NO deficiency, but not AngII, exacerbates the infil-tration of CD45 positive cells. This confirms that NO limitsinflammation,12,27,28 but contradicts studies suggestingthat inflammation is involved in the deleterious effects ofAngII on vascular function.29 However, most studiesgranting pro-inflammatory properties to AngII rely on dataderived from the use of angiotensin converting enzyme(ACE) inhibitors and/or angiotensin receptor blockers(ARBs). These drugs also have AngII independent effects,notably on endothelial cells and eNOS function,30e32 whichmay explain the discrepancy between those studies andthe present findings. Yet it is surprising that AngII had noimpact on CD45 positive cell infiltration as its deleteriouseffects are partly mediated by eNOS uncoupling and sub-sequent increase in oxidative stress and reduced NObioavailability.33,34 It is likely that the effect of AngII on theNO system in this model is not sufficient to trigger majorNO shortage, especially as NO production is known to beincreased in renin dependent models of hypertension.15

The fact that NO blockade alone triggers more CD45 pos-itive cell infiltration and IH than the AngII dependentmodels, suggests that eNOS is still functional and NO effi-cient in these models, although probably not to the level ofnormotensive animals.

Altogether the present data indicate that NO deficiencyalone is permissive to infiltration of immune cells, VSMCproliferation, and IH. However, several limitations have tobe acknowledged. The CAL model is not the best experi-mental approach for studying lumen restenosis and the roleof hemodynamic forces, because of the complete occlusionof the vessel. The partial stenosis or angioplasty models aremore clinically relevant. However, their technical complexityincreases experimental variability, limiting their widespreaduse. Nevertheless, the CAL model allows the study of earlyphenomena associated with IH, which are the cornerstonesof the restenosis process. Finally, the L-NAME treatment


results in systemic inhibition of NO production, which ismore severe than NO deficiency following vascular in-terventions. Interestingly, Havelka et al. recently reportedthat short-term balloon mediated endoluminal delivery ofNO inhibits IH following arterial injury.35 Additional researchusing mouse models of IH more comparable with humanendovascular interventions will be undertaken to differen-tiate the deleterious effects of pressure, AngII, and NO inpatients with PAD.

Significance

Today, the first line strategy to prevent restenosis afterendovascular therapy in PAD relies on drug eluting devices,being balloons or stents, coated with anti-proliferativedrugs such as paclitaxel36,37 or sirolimus.38 The mostlimiting factor of these drugs is their negative effect on re-endothelialization, leading to higher risks of thrombosis.39

Given that most vascular procedures destroy the endothe-lial inner layer of the vessel, the present study argues forthe use of NO donors following vascular intervention topalliate the lack of endogenous endothelium derived NO,thereby limiting inflammation, thrombosis, and eventuallyrestenosis. Epidemiologic evidence indicates that chronicadministration of long acting nitrates increases rather thandecreases cardiovascular events.40 In addition hypertensionis known to increase oxidative stress and NO scavenging,thereby reducing NO bioavailability.33,34 Interestingly, ACEinhibitors and/or angiotensin receptor ARBs may havebeneficial effects on eNOS function and NO bioavail-ability.30e32 The combination of AngII inhibitors and tran-sient local NO releasing strategies such as drug elutingballoons35 or stents may prove useful during endovascularprocedures to limit long-term restenosis in hypertensivePAD patients.

ACKNOWLEDGMENTS

We thank Janine Horlbeck and Jean-Christophe Stehle fortheir excellent technical assistance.

CONFLICT OF INTEREST

None.

FUNDING

This work was supported by grants from the SNF 31003A-155897, the Octav and the Marcella Botnar Foundation toJAH, the Muschamp Foundation to SD, and the SNFP1LAP3_158895 to AL.

APPENDIX A. SUPPLEMENTARY DATA

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.ejvs.2016.01.024.

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