doi:10.1152/ajpheart.01296.2006 292:2523-2531, 2007. First published Feb 2, 2007;Am J Physiol Heart Circ Physiol
Joseph L. Unthank Steven J. Miller, Laura E. Norton, Michael P. Murphy, Michael C. Dalsing andcollateral growth impairment stress in spontaneously hypertensive rat mesenteric The role of the renin-angiotensin system and oxidative
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The role of the renin-angiotensin system and oxidative stress in spontaneouslyhypertensive rat mesenteric collateral growth impairment
Steven J. Miller,1,2,3 Laura E. Norton,1 Michael P. Murphy,1,3
Michael C. Dalsing,1 and Joseph L. Unthank1,2,3
Departments of 1Surgery and 2Cellular and Integrative Physiology, Indiana University MedicalCenter, and 3Indiana Center for Vascular Biology and Medicine, Indianapolis, Indiana
Submitted 27 November 2006; accepted in final form 29 January 2007
Miller SJ, Norton LE, Murphy MP, Dalsing MC, Unthank JL.The role of the renin-angiotensin system and oxidative stress in sponta-neously hypertensive rat mesenteric collateral growth impairment. Am JPhysiol Heart Circ Physiol 292: H2523–H2531, 2007. First publishedFebruary 2, 2007; doi:10.1152/ajpheart.01296.2006.—Recent clinicaland animal studies have shown that collateral artery growth is im-paired in the presence of vascular risk factors, including hypertension.Available evidence suggests that angiotensin-converting enzyme in-hibitors (ACEI) promote collateral growth in both hypertensive hu-mans and animals; however, the specific mechanisms are not estab-lished. This study evaluated the hypothesis that collateral growthimpairment in hypertension is mediated by excess superoxide pro-duced by NAD(P)H oxidase in response to stimulation of the ANG IItype 1 receptor. After ileal artery ligation, mesenteric collateralgrowth did not occur in untreated, young, spontaneously hypertensiverats. Significant luminal expansion occurred in collaterals of sponta-neously hypertensive rats treated with the superoxide dismutase mi-metic tempol, the NAD(P)H oxidase inhibitor apocynin, and the ACEIcaptopril, but not ANG II type 1 (losartan) or type 2 (PD-123319)receptor blockers. The ACEI enalapril produced equivalent reductionof arterial pressure as captopril but did not promote luminal expan-sion. This suggests the effects of captopril on collateral growth mightresult from its antioxidant properties. RT-PCR demonstrated thatANG II type 1 receptor and angiotensinogen expression was reducedin collaterals of untreated rats. This local suppression of the reninangiotensin system provides a potential explanation for the lack ofeffect of enalapril and losartan on collateral growth. The resultsdemonstrate the capability of antioxidant therapies, including capto-pril, to reverse impaired collateral artery growth and the novel findingthat components of the local renin angiotensin system are naturallysuppressed in collaterals.
NATURAL COMPENSATION FOR PERIPHERAL arterial occlusive dis-ease includes both the formation and growth of new bloodvessels in ischemic tissues and the flow-mediated enlargementof preexisting bypass vessels. This latter process has beentermed collateral growth, arteriogenesis, or collaterogenesis.Strong evidence supports a role for angiotensin II (ANG II)signaling through the ANG II type 1 receptor (AT1R) in bothpromotion and inhibition of angiogenesis (24). Few studieshave investigated the role of the renin-angiotensin system(RAS) or ANG II in collateral growth, even though collateralgrowth provides greater hemodynamic benefit for peripheral
arterial insufficiency than capillary formation or angiogenesis(62, 69). Recent clinical and animal studies have shown thatcollateral growth is impaired in the presence of risk factorsfor vascular disease, including hypertension. Angiotensin-converting enzyme inhibitors (ACEI) have been shown topromote collateral growth in both hypertensive humans (35)and animals (17). The specific mechanisms responsible forthe impairment in hypertension and its reversal by ACEIhave not been established. Given the common use of ACEIand ANG II receptor blockers (ARB) for treatment ofvascular disease, including peripheral arterial disease, it isof critical importance to elucidate the mechanisms by whichthese drugs impact the physiological response to arterialocclusion.
We selected the spontaneously hypertensive rat (SHR) toinvestigate the mechanisms by which ACEI are able toreverse collateral growth impairment. In this animal model,vascular adaptations to arterial occlusion have been shownby two independent groups to be impaired in different organsystems: the hindlimb (16, 17) and mesentery (59). In thehindlimb, chronic administration of the ACEI ramipril in-creases tissue perfusion without altering skeletal musclecapillary or arteriole density (17). These authors speculatedthat the improved perfusion resulted from enhanced collat-eral function.
The SHR has several characteristics similar to human hy-pertension that may be involved in collateral growth impair-ment and that may be corrected by manipulation of RAS.These characteristics include endothelial dysfunction and ele-vated levels of oxidative stress and ANG II. Endothelialdysfunction or reduced nitric oxide (NO) bioavailability mayhave a primary role in the impairment of collateral growth.Several groups have shown that, during collateral growth orshear-mediated outward arterial remodeling, endothelial nitricoxide synthase (eNOS) and NO are essential for altered geneexpression/activity, matrix metalloproteinase activation, andcell recruitment (9, 34, 56, 57, 71). Excessive oxidative stresscan produce endothelial dysfunction and eNOS uncoupling.The male SHR has elevated vascular oxidant productionthroughout the vasculature (10, 53, 72). NAD(P)H oxidase is amajor, if not the predominant, source of elevated superoxide inthe SHR vascular wall (22, 60). ANG II activates NAD(P)Hoxidase activity via the AT1R (29) and expression of AT1Rand ANG II type 2 receptor (AT2R) is elevated in young maleSHR (40, 50).
Address for reprint requests and other correspondence: J. L. Unthank, Dept.of Surgery (WD OPW 425E), Indiana Univ. Medical Center, 1001 West TenthSt., Indianapolis, IN 46202-2879 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Heart Circ Physiol 292: H2523–H2531, 2007.First published February 2, 2007; doi:10.1152/ajpheart.01296.2006.
Based on these reported findings, we hypothesized that SHRcollateral growth impairment is pressure independent and me-diated by excess superoxide produced by NAD(P)H oxidase inresponse to stimulation of AT1R by ANG II. When initialresults indicated that SHR collateral growth was enhanced bythe ACEI captopril, the superoxide dismutase mimetic tempol,and the NAD(P)H oxidase inhibitor apocynin, but not by theAT1R antagonist losartan, additional studies were designed toclarify the mechanisms by which captopril had a beneficialeffect and losartan did not. The results suggest that captopril’seffect on collateral growth in SHR is mediated by its antioxi-dant properties and requires an intact NO system. In addition,there is a natural suppression of local RAS components in theshear-loaded collateral vessels.
MATERIALS AND METHODS
Animals and groups. Male SHR were obtained from Harlan (Indi-anapolis, IN) and studied at �10 wk of age. All procedures wereapproved by the School of Medicine Institutional Animal Care andUse Committee. Rats were randomly designated for study of collateralgrowth or for telemetric measurement of blood pressure. Subgroupsreceived different drug therapy. Captopril and apocynin were obtainedfrom Fisher Scientific; tempol, NG-nitro-L-arginine methyl ester(L-NAME), and enalapril were obtained from Sigma. PD-123319 andlosartan were gifts from Pfizer and Merck, respectively. All drugs
were given in drinking water, except for PD-123319, which wasadministered via osmotic minipump (Alzet; model 2ML2). Drugdelivery methods and concentrations/doses were based on publishedstudies that have demonstrated effectiveness in reducing oxidativestress (6, 51, 66, 68) or affecting arterial or ventricular remodeling (7,14, 28, 32, 41, 46). Concentrations in drinking water (mM) were 9.2captopril, 1.0 and 5.0 tempol, 1.5 and 3.0 apocynin, 0.2 and 0.6losartan, and 0.6 enalapril. In some experiments, L-NAME (0.1 mM)was added along with captopril in drinking water. For delivery ofPD-123319 (�30 mg �kg�1 �day�1), the osmotic minipump was pre-filled with PD-123319 (62.5 mg/ml) under sterile conditions beforeimplantation. After anesthesia and with aseptic technique, theminipump was placed in a subcutaneous pocket in the nape of theneck.
Model of collateral growth and its assessment. We utilized ourmodel of mesenteric artery collateral growth, as previously described(59). Advantages of this model over the femoral artery ligation modelinclude the clearly defined collateral pathway and the ability to makerepeated measurements of in vivo arterial diameters. Tuttle et al. (59)have previously demonstrated with this model that collateral growthoccurs in normotensive control animals, but not SHR. Surgical depthanesthesia was achieved with isoflurane, and an antibiotic was admin-istered (cephazolin, 15 mg/kg sc). Then, with the use of aseptictechnique, a laparotomy was performed, and the terminal ileumexteriorized into a heated tissue support chamber. The bowel andmesentery were immersed in phosphate-buffered saline or coveredwith plastic wrap at all times. As illustrated in Fig. 1A, several
Fig. 1. Mesenteric model of collateral arterygrowth. A: a region of the terminal ileum isselected so that ligation of 3–4 sequentialileal arteries will create a collateral-depen-dent pathway with 40–50 microvascularperfusion units (first-order arterioles) be-tween the two collateral vessels. Two arter-ies are also selected as same-animal con-trols. Great care is taken to ligate only thearteries, leaving companion veins intact.Digital images are then made at specificlocations on each control and collateral ar-tery under dilated conditions and repeated 7days later. Paired images from each artery asdepicted in B are later used for inner diam-eter measurement (red cell column) and cal-culation of percent change in inner diameter.
H2524 OXIDANT STRESS AND SHR COLLATERAL GROWTH IMPAIRMENT
sequential ileal arteries were ligated with 9-0 monofilament suture,such that a region of bowel containing �45 microvascular perfusionunits was dependent on collateral arteries for perfusion. The suffusionsolution was then replaced with one containing 1.0 mM adenosine and0.1 mM sodium nitroprusside to dilate the vasculature. Digital imagesof collateral and same-animal control arteries were acquired at max-imum magnification with a dissecting microscope (Leica MZ 9.5) andcamera (Spot Insight 4 Firewire). The bowel was returned to theabdominal cavity, and the incision was closed in two layers with 4-0suture. Pain medication was administered for 2 days (buprinorphine0.01 mg/kg sc bid). One week later, laparotomy and acquisition ofdigital images were repeated. Representative digital images are shownin Fig. 1B. The red cell column in arteries was measured as innerarterial diameter with image analysis software (Image-J), and thepercent change in diameter was calculated. For assessment of collat-eral growth, drug therapy was initiated 3-5 days before model creationand continued until final experimentation.
Telemetry measurements of arterial pressure. Under general in-haled anesthesia, the right groin was shaved and prepped. Usingaseptic technique, a 2-cm incision was made, and blunt forceps wereused to expose the femoral artery, vein, and nerve. The femoral arterywas carefully isolated from the vein and nerve, ligated distally with4-0 silk suture, and clamped proximally with a microvascular clamp.A small arteriotomy was created in the femoral artery. The catheter ofthe TA11PA-C40 transmitter (Data Sciences International, St. Paul,MN) was introduced through this arteriotomy. The catheter wasadvanced antegrade �5 cm such that the tip of the catheter rested inthe abdominal aorta. Another 4-0 silk suture was tied around thefemoral artery, just proximal to the entrance of the catheter, to securethe catheter in place. Blunt dissection through the same incision wasused to create a subcutaneous pocket in the flank for housing of thetransmitter. The transmitter was placed, and the subcutaneous layerwas closed to seal the pocket using a running 4-0 Polysorb suture. Theskin incision then was closed. Animals were allowed to recover for2–3 days, at which time initial 24-h recordings of heart rate, systolic,mean, and diastolic blood pressure were obtained, utilizing DSIreceivers and software. A second set of recordings were obtained 3–4days after animals were started on drug therapy. The third (final) setof recordings was obtained 7–10 days after initiation of drug therapy.
Quantitative RT-PCR. Relative differences in mRNA expressionwere determined by using real-time quantitative PCR. For vesselisolation, the abdominal aorta of an anesthetized rat was cannulatedabove the iliac bifurcation. After ligating both renal arteries and theproximal aorta, the mesenteric circulation was perfused with 30 ml ofcold, phosphate-buffered saline followed by 10 ml of RNAlater(Ambion, Austin, TX). Isolated mesenteric arteries were excised andpreserved in RNAlater at �20°C before RNA isolation. Tissues wereweighed, disrupted by using a bead homogenizer (FastPrep System;QBIOgene, Carlsbad, CA), and total RNA was purified by using anRNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA). To ensurehigh-quality RNA, sample concentration and integrity were deter-mined by using 260- to 280-nm absorbance ratio and by analysis withan Agilent 2100 Bioanalyzer (RNA 6000 Nano Chip Kit). Aliquots ofpurified total RNA (0.5 �g) were treated to remove contaminatinggenomic DNA (DNA-free; Ambion) and then reverse transcribedusing Ready-To-Go You Prime First Strand Beads (GE Healthcare/Amersham Biosciences, Piscataway, NJ) with random decamer prim-ing. For PCR, an aliquot of cDNA (5.0 �l of 1:5 to 1:50 dilution) wascombined with the appropriate primers for the target or �-actinendogenous control (TaqMan Gene Expression Assays; Applied Bio-systems, Foster City, CA) in the presence of PCR reagents (QuantiTectProbe PCR Kit; Qiagen). Reactions were run in triplicate on anApplied Biosystems 7500 Real-Time PCR System using relativequantification (ddCT, where CT is cycle threshold) with dual-labeled(FAM/MGB) probes as the product detection method. Standard 7500PCR cycling conditions were used for angiotensinogen (Agt), butmodified to 45 cycles for AT1R experiments. Differences in PCR
product yields between groups were determined by comparing thefold differences between target mRNA after normalization to �-actin.
Statistical analyses. Tests for statistical significance were per-formed with SigmaStat 3.0. Repeated-measures ANOVA was usedwith multiple pairwise comparisons performed with the Holm-Sidakmethod. Data are presented as group averages with SE of the mean.The correlation between changes in collateral artery diameters andarterial pressure was evaluated with linear regression analysis.
RESULTS
Captopril reverses the impairment of collateral growth. Themaximally dilated inner diameters of control and collateralarteries were similar within groups, averaging 267 � 5.1 �m atinitial model creation. Measurements of the same vessel diam-eters from time of ligation to 1 wk later were made for controland collateral arteries in all groups. Changes in maximallydilated inner diameters for the various treatment groups arereported as percent diameter change in Fig. 2. No significantincreases occurred in the control arteries of any group. Thediameters of the collateral arteries in untreated (control) SHRwere not increased, confirming our laboratory’s earlier study ofcollateral growth in SHR (59). In captopril-treated SHR, aremarkable increase (141 � 13.6 �m; P � 0.001) occurred incollaterals.
Tempol and apocynin, but not losartan, enhance collateralgrowth. To investigate the mechanisms by which captoprilreversed SHR collateral growth impairment, the effect ofcommonly used chronic doses of tempol, apocynin, and losar-tan were evaluated for their effect on collateral growth. Theresults are summarized in Fig. 2B. Statistically significantenlargement of the collateral arteries did not occur for any ofthese groups at the initial dose tested. There was, however, atendency for collateral diameter enlargement with both tempoland apocynin (29 � 13 and 39 � 15 �m increase, respec-tively). Subsequent experiments were performed with in-creased concentrations (5 mM for tempol, 3 mM for apocynin).These higher concentrations resulted in statistically significantincreases in collateral diameters, 88 � 14 (P � 0.006) and77 � 14 �m (P � 0.001), respectively, for tempol andapocynin. These diameter increases were not statistically dif-ferent from those observed with captopril. A higher dose oflosartan was also tested, but did not promote collateral growth.
Enalapril and PD-123319 do not promote collateral growth;L-NAME inhibits captopril’s enhancement of collateral growth.After the unexpected results with losartan, additional experi-ments were performed to clarify the mechanisms by whichcaptopril, but not losartan, promoted collateral growth. ACEIcan influence remodeling events by their effect on the kal-likrein-kinin system and stimulation of NO production (15).The sulfhydryl group within captopril endows it with antioxi-dant properties (38) not found in most ACEI. The AT2R mayalso impact vascular structure and remodeling (32). To addressthese possibilities, we investigated the effect on collateralgrowth of enalapril as an alternative ACEI without the antiox-idant potential of captopril (38, 52), the selective AT2R antag-onist PD-123319, and low-dose L-NAME in combination withcaptopril. The results are presented in Fig. 2C. None of thesetreatments resulted in significant collateral growth. The lack ofan enalapril effect suggests that captopril’s enhancement ofcollateral growth is not solely mediated by inhibition of an-giotensin-converting enzyme. The absence of an effect of
H2525OXIDANT STRESS AND SHR COLLATERAL GROWTH IMPAIRMENT
PD-123319 at a dose previously shown to prevent ANG II-induced AT2R-dependent vascular remodeling (32) indicatesthat the beneficial effect of captopril is not mediated throughthe AT2R. The prevention of captopril’s stimulation of collat-eral growth by low-dose L-NAME indicates that captopril’saction is mediated by or at least requires NO, consistent withthe mechanisms by which captopril has been shown to inhibitintimal hyperplasia (15).
Collateral growth enhancement is independent of arterialpressure reduction. Arterial pressures were measured by te-lemetry in separate animals receiving identical drug treatments.This was done primarily to determine the effect of bloodpressure reduction on collateral growth. Hypertension itselfcan induce superoxide production, medial hypertrophy, and
inward remodeling, which could reduce the capacity for flow-induced outward remodeling. A secondary objective was toverify that the doses of enalapril and losartan, which had noeffect on collateral growth, were indeed effective in reducingarterial pressure. Average 24-h arterial pressure in younguntreated SHR was 135 � 1.9 mmHg. Captopril and enalaprilproduced equivalent reductions in arterial pressure (30 � 1.1vs. 28 � 2.6%). Both doses of losartan produced similarpressure reductions, averaging 16 � 2.5%. Mean arterial pres-sure was also significantly reduced by the highest dose oftempol. The pressure reductions obtained with losartan andtempol were less than captopril. Treatment with the higherdose of apocynin did not significantly decrease arterial pres-sure (4 � 0.7%). The potential correlation between pressurereduction and collateral growth was evaluated with regres-sion analysis of changes in arterial pressure and collateralgrowth (Fig. 3).
The local RAS system is suppressed in SHR collaterals. Withthe understanding that ANG II and AT1R levels and NAD(P)Hoxidase expression are increased in the young SHR (6, 40, 47,50) and that ANG II activates NAD(P)H oxidase via the AT1R(29), we were without an explanation for why blockade of theAT1R by losartan did not promote collateral growth. Inspec-tion of preliminary genomic data from our laboratory obtainedwith microarray analysis revealed a trend for some componentsof the RAS to be downregulated in SHR collaterals. Since thiscould potentially explain the lack of an effect of losartan aswell as enalapril on collateral growth, RT-PCR experimentswere performed 1 and 3 days after arterial ligation toevaluate AT1R and Agt mRNA expression. By day 3 in ourmodel, cell proliferation had begun and luminal expansionwas occurring (57). Collateral arteries were compared withthe same animal control vessels. The data indicate thatexpression levels of both molecules were consistently re-duced in collaterals (Fig. 4).
Fig. 3. Correlation of the increase in collateral diameter with decrease in meanarterial pressure (MAP). Regression analysis was performed from averagesobtained for collateral growth (CG) and blood pressure measurement bytelemetry in different groups of animals and for various drugs. The slope of theregression line was not significantly different than zero (P � 0.222), and thecorrelation coefficient was very low (0.007). The results with enalapril dem-onstrate that pressure reduction alone is not sufficient to stimulate collateralgrowth. In addition, the data for apocynin establish that collateral growth canoccur in the presence of hypertension.
Fig. 2. Effect of drug treatment on collateral growth. A–C: the percent changein maximally dilated in vivo inner diameter of control and collateral arteries forthe various treatments is shown. During the week between measurements,there were no significant changes in diameter of control arteries. The diameterof collaterals was increased only after treatment with captopril, tempol, andapocynin. N � 4–6.
H2526 OXIDANT STRESS AND SHR COLLATERAL GROWTH IMPAIRMENT
This study was performed in an animal model of vasculardisease characterized by impaired collateral artery growth. Toour knowledge, these results are the first to demonstrate thecapability of antioxidant therapy to reverse impaired collateralgrowth in a model of endothelial dysfunction. Because all ofthe conditions that are associated with impaired collateralgrowth (hypertension, diabetes, hypercholesterolemia, and ag-ing) in patients (1, 5, 23, 27, 37) and animals (11, 44, 45, 49,58, 59, 63) are associated with elevated oxidative stress, webelieve that this is an important finding. In addition, a novelobservation is the suppression of local RAS components indeveloping collateral vessels.
In hypertension, oxidative stress may be increased by mul-tiple enzyme systems, including xanthine oxidase, uncoupledeNOS, the mitochondrial respiratory chain, and NAD(P)Hoxidase (30). In male SHR, superoxide production by vascularNAD(P)H oxidase is increased (22, 60). NAD(P)H oxidaseexpression/activation is regulated by many factors, includingendothelin and angiotensin. ANG II vascular levels may be10� greater than in plasma in normotensive rats and evenhigher in SHR (3). Activation of the AT1R by ANG II resultsin activation of NAD(P)H oxidase and elevation of superoxideformation (29). Captopril, at a comparable dose used in thisstudy, reduces SHR arterial pressures, plasma angiotensin andendothelin, and oxidative stress (6). Our initial results con-firmed earlier studies of impaired adaptation to arterial occlu-sion in SHR (16, 17, 59) and demonstrated a remarkable effectof captopril on collateral growth (51 � 8.7% luminal expan-sion in 7 days, Fig. 2A). The collateral growth observed withcaptopril treatment was equivalent to or greater than what wehave previously observed in young normotensive rats (59, 61).These results, together with the earlier report demonstratingthat ramipril improves perfusion in the hindlimb ligation model(17), clearly establish the therapeutic capacity of ACEI topromote vascular compensation in the SHR. The lack of anincrease in capillary or arteriolar density in the earlier reportand the clear demonstration of luminal expansion of preexist-ing bypass vessels in the present study suggest that the ACEI
enhancement involves collateralization of existing vesselsrather than angiogenesis or neovascularization. A similar ob-servation with ACEI has been reported for patients with cor-onary artery disease (35). Based on the known phenotype ofSHR and the effects of captopril, we hypothesized that SHRcollateral growth impairment is mediated by excess superoxideproduced by NAD(P)H oxidase in response to stimulation ofthe AT1R by ANG II. Because pressure elevation can increaseoxidant production (39) and initiate medial hypertrophy andinward remodeling in resistance vessels (73), we were alsointerested in determining whether pressure reduction was im-portant for the outward remodeling associated with collateralgrowth.
The results are consistent with our hypothesis regarding arole for excess superoxide produced by NAD(P)H oxidase inSHR collateral growth impairment. Chronic administration ofthe SOD mimetic tempol restored the capacity for collateralgrowth in a dose-dependent manner (Fig. 2B). Other studieshave shown chronic administration of tempol to SHR reducesoxidative stress, arterial pressure, and microvascular rarefac-tion and increases NO bioavailability (28, 51, 66, 68). Apoc-ynin is a widely used inhibitor of NAD(P)H oxidase. In vivostudies with chronic apocynin administration (1.5–2.5 mM indrinking water) have shown decreased oxidative stress in SHR(41) and attenuation of aldosterone-induced elevation ofNAD(P)H oxidase, arterial pressure, p22phox expression, andcardiac remodeling (42). In our study, apocynin also promotedcollateral growth in a dose-dependent manner (Fig. 2B), im-plicating a significant role of NAD(P)H oxidase in the impair-ment of SHR collateral growth. These results suggest thatantioxidant therapy might be effective as either primary oradjuvant therapy to improve vascular compensation to arte-rial insufficiency, specifically collateral growth. While com-pleted clinical trials with antioxidants have revealed nosignificant benefit related to cardiovascular health, signifi-cant flaws of these trials have been identified (21, 55). Theseshortcomings include the lack of prescreening to identifypatients with elevated oxidative stress and the utilization ofineffective antioxidant therapies, such as vitamins C and E.
Unlike the results with tempol and apocynin, the effects oflosartan and enalapril (Fig. 2) were the opposite of what weanticipated. Neither was effective in promoting collateralgrowth. In contrast, the previously cited study in the SHRhindlimb (17) demonstrated improved perfusion after femoralartery ligation in response to ramipril, a nonsulfhydral ACEI.In addition, the clinical study that demonstrated significantbenefit of ACEI related to collateral growth in patients withcoronary artery disease excluded patients receiving captopril(35). There are several potential explanations for these dispar-ities. In the ramipril study (17), therapy was administeredimmediately after rather than before arterial ligation and wascontinued for 28 days vs. 7 days in the present study. It ispossible that longer term therapy with losartan and enalaprilwould have been effective in our study. However, other inves-tigations have revealed differences between various ACEI andARBs for specific outcomes (4, 20). Zofenopril, another sulf-hydryl ACEI, but not enalapril, reduced oxidative stress inpatients with essential hypertension (38). However, other stud-ies, including one in SHR (33), have shown enalapril to beeffective in reducing oxidative stress. In a rabbit hindlimbischemia model, quinaprilat was much more effective in pro-
Fig. 4. mRNA expression of angiotensin II type 1 receptor (AT1R) andangiotensinogen (Agt). Relative mRNA expression of AT1R and Agt wasdetermined by real-time RT-PCR for whole vessel lysates of control andcollateral arteries and normalized to �-actin. The relative expression in thecollateral was then normalized to the same-animal control, and the means andSEs are reported in the figure. The 1- and 3-day time points were evaluated todetermine whether expression was altered before and after the collateral hadbegun to expand (57). N � 4 for 1- and 3-day time points.
H2527OXIDANT STRESS AND SHR COLLATERAL GROWTH IMPAIRMENT
moting angiogenesis and inhibiting tissue ACE than captopril(18). In experimental tumor growth, captopril, but not lisinoprilor enalaprilat, inhibits angiogenesis (64). Such findings may berelated to differences in ACEI properties [e.g., potency, bio-availability, distribution and affinity for tissue ACE (8), alteredreceptor distributions (31), and/or changes in levels of ANG IIand ANG(1-7) (19, 25)]. These diverse outcomes for differentRAS suppressing drugs illustrate the need for additional study.
Another unexpected observation was the suppression of thelocal RAS in mesenteric collaterals (Fig. 4). This novel findingof reduced mRNA levels of AT1R and Agt is consistent withprevious studies, which have shown the existence of a vascularRAS and the downregulation of RAS components by shearstress and/or NO. Functional evidence of a local microvascularRAS was reported in 1994 by Vicaut and Hou (63a). In anisolated cremaster preparation, greater arteriolar vasoconstric-tion to Agt and ANG I was observed in SHR than in WistarKyoto rats, and this vasoconstriction was prevented by ACEI.More recently, Agoudemos and Greene (3) have demonstrated
the presence of Agt and renin mRNA and protein in rat skeletalmuscle arterioles. Tissue ANG II levels were more than 10�greater than plasma levels in normotensive animals and evenhigher in SHR (3). Greene’s group has also shown that shearstress elevation alters endothelial cell function by suppressingACE gene and protein expression in vitro and in vivo (43).More recent studies by other groups indicate that both ACEand additional RAS components are downregulated by shearstress or NO. Miyakawa et al. (36) found two novel shear stressresponse elements in the rat ACE promoter, which downregu-late ACE expression when shear is elevated. A flow or shearstress-dependent suppression of AT1R is also consistent withthe finding of Adams et al. (2) that physical exercise isassociated with downregulation of this receptor in humaninternal mammary arteries. Yan et al. (67) have reviewedmechanisms by which NO may regulate the ANG II signalingpathway, including reduction of ACE activity and AT1R ex-pression. Thus evidence exists in other systems and experi-mental conditions to support the hypothesis that elevated shear
Fig. 5. Proposed interactions between the renin angiotensin system, NADPH oxidase, oxidative stress, and nitric oxide (NO) bioavailability that influencecollateral growth. Our central hypothesis has been that the balance between NO and superoxide levels determines in large part whether collateral growth issuccessful or impaired. Local levels of NO and O2
� can impact collateral growth by altering the expression and activity of growth modulators and matrixmetalloproteases and function of bone marrow-derived cells. In the spontaneously hypertensive rat and other models of endothelial dysfunction characterized byelevated angiotensin II and/or NAD(P)H oxidase, elevated levels of superoxide or daughter compounds can impair growth through multiple mechanisms. In thisschematic, solid and dashed arrows represent stimulation and inhibition, respectively. The schematic emphasizes how elevated shear and/or NO production incollateral vessels may suppress components of the local renin angiotensin system. eNOS, endothelial NO synthase; ACE, angiotensin-converting enzyme; AT1and AT2, angiotensin II receptor types 1 and 2, respectively.
H2528 OXIDANT STRESS AND SHR COLLATERAL GROWTH IMPAIRMENT
stress and/or NO suppresses the local vascular RAS. However,additional studies are needed to confirm the functional rele-vance of the mRNA suppression and establish its role oncollateral growth in this and other models.
The results suggest that suppression of the local RAS,together with reduction of oxidative stress in SHR, are essen-tial for the successful outward remodeling of collateral vessels.The inward remodeling of mesenteric resistance arteries inyoung SHR is prevented or reversed by chronic suppression ofthe RAS with enalapril and losartan (46). Our observations ofreduced Agt and AT1R expression in SHR collaterals supportthis hypothesis and also provide a potential explanation for thesystemic effect of enalapril and losartan on arterial pressurewithout a beneficial effect on collateral growth.
Although ACEI and ARB are widely prescribed for patientswith vascular disease, the specific role of the vascular RAS intumor angiogenesis (13, 64) and adaptative responses to arte-rial insufficiency (12, 17, 18, 35, 48, 54) remain controversial.It is clear from existing studies that there are multiple mech-anisms/pathways through which RAS may promote and sup-press vascular remodeling. Even within different vascular bedsof the same animal, RAS-suppressing drugs can have opposingeffects (12). Recent studies have shown that ANG(1-7) mayoppose actions of ANG II. Also, the hematopoietic stem cellregulator N-acetyl-seryl-aspartyl-lysyl-proline promotes angio-genesis (65) and is modulated by ACEI, including captopril(26). These recent observations, together with contradictorystudies, emphasize the need for additional studies to clarifymechanisms by which angiotensin and RAS-suppressing drugsinfluence vascular remodeling, including that which occurs inresponse to arterial occlusion.
While we believe this study has potentially important clin-ical significance, there are a number of limitations with ourstudy that should be considered. First, therapy was initiatedbefore arterial insufficiency was created. This approach wastaken, because our objective was to determine the mechanismsresponsible for collateral growth impairment in young SHRrather than ways to promote collateral growth. While theprevious work by Emanueli et al. (17) demonstrates thattherapy can be effective when given immediately after abruptligation, future work is needed to determine whether similartherapies can reverse a longstanding impaired response. Asalready indicated, our treatment duration was short term. Othertherapies may be just as effective when administered for alonger duration. Also, we studied young rats during the devel-opmental stage of hypertension. We anticipate that therapywould be effective in established hypertension, but additionalpathology may develop with aging and long-term hyperten-sion, which may reduce or limit effectiveness. It is alsopossible that responses in mesenteric collaterals do not reflectwhat occurs in other organs. However, our results are consis-tent with studies that have shown ACEI to increase hindlimbperfusion after arterial ligation (17, 70) without stimulatingangiogenesis (17).
In conclusion, we have demonstrated that impaired collateralgrowth in SHR can be dramatically reversed with antioxidanttherapy. The results are consistent with the hypothesis thatelevated NO levels are required for successful collateralgrowth and that excess superoxide produced from NAD(P)Hoxidase impairs collateral growth by suppressing bioavailableNO. Downregulation of the local RAS may also be important
for collateral growth. A schematic is presented in Fig. 5 toillustrate how these components may interact to influencecollateral growth. The role of the RAS in angiogenesis andarteriogenesis is controversial, and the results from this studyraise questions about the importance of suppressing the RAS topromote collateral growth. Given the widespread use of ACEIand ARB, this remains an important area for investigation.
ACKNOWLEDGMENTS
The excellent technical assistance from Randal G. Bills and P. MelaniePride is gratefully acknowledged, as is the proof editing by Tonia Miller.
GRANTS
This work was supported by National Heart, Lung, and Blood InstituteGrant HL-42898.
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