ACE2 Activation Promotes Antithrombotic Activity

2 1 0 | F R A G A - S I L V A E T A L . | M O L M E D 1 6 ( 5 - 6 ) 2 1 0 - 2 1 5 , M A Y - J U N E 2 0 1 0

INTRODUCTIONThrombogenic events, such as ische-

mic stroke, pulmonary embolism, deepvenous thrombosis, mesenteric ische-mia and acute coronary syndrome, aremajor complications of certain patho-logical conditions, such as hyperten-sion, atherosclerosis and diabetes melli-tus (1). Despite the many therapeuticadvances and increasingly effectivedrug treatments, thrombogenic eventsremain the major cause of morbidityand mortality worldwide (2). Signifi-cant efforts have been made to identifynovel drug treatments.

The renin-angiotensin system (RAS) isa pivotal regulator of cardiovascularfunction. Increased activity of the

vasoconstrictive and proliferative axis(angiotensin [Ang] II/angiotensin- converting enzyme [ACE]/AT1 receptor)has been reported to be associated with ahigher risk of acute thrombosis throughthe destabilizing of atheroscleroticplaque and enhancing of platelet activityand coagulation (3). In the classical con-cept, the RAS is made up of a series ofenzyme reactions that culminate in thegeneration of Ang II, the main peptide ofthis system, by ACE. Ang II has manyadverse cardiovascular effects when act-ing through the AT1 receptor. However,this simplistic concept of a linear RAScascade has given way to a more com-plex concept since the discovery ofACE2, which is an ACE homologue (4,5).

Unlike ACE, ACE2 catalyzes the conver-sion of Ang II to the hepta peptide Ang-(1-7) (6). Ang-(1-7) has been reported tobe counterregulatory to Ang II actions,promoting many beneficial cardiovascu-lar effects through the interaction with itsown receptor Mas (7). Therefore, a novelconcept of two opposing axes regulatingthe activity of the RAS has emerged: theACE/Ang II/AT1R axis is involved inhypertensive, proliferative, vasoconstric-tive, hypertrophic and fibrotic actions;whereas the counterregulatory ACE2/Ang-(1-7)/ Mas axis is involved in vaso-protective actions of the RAS (5,8).

Given these recent advances, it maybe hypothesized that the ACE2/Ang-(1-7)/Mas axis is involved in the hemo-static process, and its activation pro-duces antithrombotic activity. The aimof the present study was to evaluatethe role of ACE2 in thrombus forma-tion in normotensive and hypertensiverats and investigate the effects onthrombus formation of a novel ACE2activator (XNT) (9).

ACE2 Activation Promotes Antithrombotic Activity

Rodrigo A Fraga-Silva,1 Brian S Sorg,2 Mamta Wankhede,2 Casey deDeugd,2 Joo Y Jun,1 Matthew B Baker,3Yan Li,3 Ronald K Castellano,3 Michael J Katovich,4 Mohan K Raizada,1 and Anderson J Ferreira1,5

Departments of 1Physiology and Functional Genomics, 2Biomedical Engineering, 3Chemistry, and 4Pharmacodynamics, University ofFlorida, Gainesville, Florida, United States of America; 5Department of Morphology, Biological Sciences Institute, Federal Universityof Minas Gerais, Belo Horizonte, Brazil

The aim of the present study was to test the hypothesis that the activation of the angiotensin-converting enzyme(ACE)2/angiotensin-(1-7)/Mas receptor axis by use of a novel ACE2 activator (XNT) would protect against thrombosis. Thrombiwere induced in the vena cava of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, and ACE2 and ACE activ-ity in the thrombus was determined. Real-time thrombus formation was viewed through intravital microscopy of vessels in nudemice. Thrombus weight was 40% greater in the SHR (4.99 ± 0.39 versus 7.04 ± 0.66 mg). This weight increase was associated with a20% decrease in ACE2 activity in the thrombus. In contrast, there were no differences between the WKY and SHR in ACE2 proteinand ACE activity in the thrombi. ACE2 inhibition (DX600; 0.1 µmol/L/kg) increased thrombus weight by 30% and XNT treatment(10 mg/kg) resulted in a 30% attenuation of thrombus formation in the SHR. Moreover, XNT reduced platelet attachment to injuredvessels, reduced thrombus size, and prolonged the time for complete vessel occlusion in mice. Thus, a decrease in thrombus ACE2activity is associated with increased thrombus formation in SHR. Furthermore, ACE2 activation attenuates thrombus formation andreduces platelet attachment to vessels. These results suggest that ACE2 could be a novel target for the treatment of thrombo-genic diseases.© 2010 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2009.00160

Address correspondence and reprint requests to Mohan K Raizada, Department ofPhysiology and Functional Genomics, College of Medicine, University of Florida, PO Box 100274, Gainesville, FL 32610. Phone: (352) 392-9299; Fax: (352) 294-0191; E-mail:[email protected] November 3, 2009; Accepted for publication January 21, 2010; Epub(www.molmed.org) ahead of print January 22, 2010.

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MATERIALS AND METHODS

AnimalsAll procedures involving animals were

carried out in compliance with approvedInternational Animal Care and Use Com-mittee protocols and University of Flor-ida regulations. Male Wistar Kyoto(WKY) rats and spontaneously hyperten-sive rats (SHR) 12- to 13-wks-old (bodyweight: 280 to 310 g) were purchasedfrom the Charles River Laboratories(Wilmington, MA, USA). Nude malemice 9- to 10-wks-old were purchasedfrom Harlan Sprague Dawley Inc. (Indi-anapolis, IN, USA).

Thrombosis Model in RatsThrombus formation was induced in

the abdominal vena cava of SHR andWKY using a ferric chloride (FeCl3) solu-tion, as described elsewhere (10). Briefly,after anesthesia (ketamine, xylazine andacepromazine at 30, 6, and 1 mg/kg, re-spectively), the jugular vein was cannu-lated for drug administration. Subse-quently, the abdominal vena cava wasexposed via a midline abdominal inci-sion and was carefully separated fromthe surrounding tissues. Either XNT at 10 mg/kg, the ACE2 inhibitor DX600 at0.1 µmol/L/kg, or vehicle (saline) wasadministered 5 min before thrombus in-duction. The XNT and DX600 doses werebased on previous studies (9,11). Filterpaper (3 × 5 mm) steeped in a 5% FeCl3solution was topically applied to thevena cava 5 mm below the left renalvein. The paper was removed after 3 minand the abdomen closed with suture.After 30 min, the thrombus formed wascarefully removed, weighed and frozenat –80°C. The thrombus was thenprocessed for ACE and ACE2 activityand Western blot analysis.

Determination of ACE and ACE2Activity

ACE and ACE2 activity was deter-mined in the thrombi obtained from theWKY and SHR by using their respectivefluorogenic peptide substrates (ACE sub-strate, fluorogenic peptide V [FPS V],

Mca RPPGFSAFK(Dnp)-OH, catalog. ID:ES005; ACE2 substrate, fluorogenic pep-tide VI [FPS VI], Mca-YVADAPK(Dnp)-OH, catalog. ID: ES007, where Mca is7-(methoxycoumarin-4-yl) acetyl and Dnpis 2,4-dinitrophenyl; R&D Systems, Min-neapolis, MN, USA), as described else-where (12). The reaction buffer (75 mmol/LTris/HCl [pH 7.5], 1 mol/L NaCl and 0.5 mmol/L ZnCl2) was added to thethrombus and sonicated; the protein con-tent was determined by use of a stan-dard Bradford assay. The enzymes re-move the C-terminal dinitrophenylmoiety that quenches the inherent fluo-rescence of the 7-methoxycoumaringroup, resulting in an increase in fluores-cence at excitation and emission spectraof 328 and 392 nm, respectively. Enzymeactivity was determined using a SpectraMax Gemini EM Fluorescence Reader(Molecular Devices, Sunnyvale, CA,USA). All assays were performed at leastin triplicate and samples were read everymin for at least 120 min immediatelyafter the addition of fluorogenic peptidesubstrates at 37°C. Enzyme activity wascorrected to the background and the datawere normalized considering untreatedWKY as having 100% ACE2 activity.

Western Blotting for ACE2Sixty micrograms of protein extracted

from thrombi of WKY rats and SHR wererun on a 12% SDS-PAGE gel. The pro-teins were then transferred onto a nitro-cellulose membrane. The membrane wasprobed with the rabbit polyclonal anti-body against ACE2 (sc-20998 [Santa CruzBiotechnology, Santa Cruz, CA, USA]1 µg/mL in 1% milk/Tris-bufferedsaline-Tween solution) overnight at 4ºC.Membrane was washed three times for10 min in Tris-buffered saline–Tween so-lution and incubated with antirabbitIgG–horseradish peroxidase–conjugatedsecondary antibody (1:5000) for 1 h atroom temperature. After the finalwashes, the membrane was incubatedwith chemiluminescent agent for 1 minand then exposed to a film to visualizethe protein bands. β-actin was used as aloading control.

Intravital MicroscopyReal-time thrombus formation was

visualized using a mouse dorsal skin-fold chamber and intravital microscopyin nude mice, as described elsewhere(13,14). Briefly, the mice were anaes-thetized with a mixture of ketamine(100 mg/kg) and xylazine (10 mg/kg)intraperitoneally and the jugular veinwas cannulated for intravenous access.A titanium window chamber was surgi-cally implanted in the dorsal skin flapof the mice. From the extended doublelayer of the dorsal skin, the top layerwas removed to visualize the vascula-ture on the opposite side of the skinflap in a circular area of about 6 mm2.Veins with a diameter of 80 to 100 µmwere chosen for visualization of local-ized thrombus formation via intravitalmicroscopy. The cannulated jugularvein was used to treat the mice with in-travenous carboxyfluorescein diacetatesuccinimidyl ester (Invitrogen, Carls-bad, CA, USA; cat# C1157), which is anonfluorescent precursor taken up intoplatelets and partly into leukocytes,which forms a stable fluorochrome de-nominated carboxyfluorescein succin-imidyl ester (CFSE) in the cells. CFSE-labeled platelets flowing through themouse vasculature and the subsequentvascular injury–induced thrombus formation were visualized through multifunctional intravital fluorescencemicroscopy. A Zeiss AxioImager micro-scope (Carl Zeiss, Thornwood, NY,USA) served as the basic platform. Fluorescently labeled platelets in themouse vasculature were imaged at amagnification of 10× using the EC Plan-NeoFluar long- working distance objec-tive (Carl Zeiss). An ANDOR iXon elec-tron multiplying CCD (EMCCD)camera (ANDOR Technology, SouthWindsor, CT, USA) was used to capturestreaming videos of the fluorescently la-beled platelets in the microvasculatureat a frame rate of about 30 Hz. A ZeissFluoArc mercury lamp was used as thelight source for the fluorescent imaging.Fluorescent platelets were viewed usingan FITC filter set (Carl Zeiss; filter set


A N T I T H R O M B O T I C E F F E C T O F A C E 2

10, excitation: BP 450 to 490; emission:BP 515 to 565). The appropriate vesselregion in the mouse vasculature was se-lected for thrombus induction based onvessel size (80 to 100 µm) and propernetwork functionality. Either XNT at5 mg/kg or vehicle (saline) was admin-istrated 5 min before thrombus induc-tion. Vascular injury and thrombus for-mation were induced by a precisetopical application of filter paper (3 mm2)soaked in 5% FeCl3 solution on the se-lected vessel location. After 2 min, thepaper was removed and the vesselswere washed with saline at 37°C.Streaming videos of 20-s duration wereobtained for the treatment area everymin. The microvessels were viewed approximately 30 min after FeCl3 appli-cation, and during this period, an eval-uation was performed of the time nec-essary to stop the blood flow as a resultof the total occlusion of the vessel andthe thrombus size over time.

Statistical AnalysisAll data are expressed as mean ± SEM.

Comparisons between experimental andcontrol groups were analyzed using one-or two-way ANOVA followed by theBonferroni posttest or Student t test. Dif-ferences were considered significant at aP ≤ 0.05.

RESULTS

Higher Thrombus Formation in SHR IsAssociated with Lower ACE2 Activity

As expected, FeCl3-induced thrombusweight was 40% higher in the SHR com-pared with the WKY (4.99 ±0.39 versus7.04 ±0.66 mg in SHR) (Figure 1A). Thisresult was associated with a 20% reduc-tion in ACE2 activity in the thrombifrom the SHR (Figure 1B). There wereno differences in ACE2 protein levels inthe thrombi from the SHR and WKY(Figure 1C, D). These data reveal thatthe greater thrombus formation in the

SHR was associated with lower ACE2activity, with no significant change inACE2 protein. In contrast with ACE2,there were no significant differences inACE activity in the thrombi between theSHR and WKY (Figure 2A). However,the ratio between ACE2 and ACE activ-ity was significantly decreased in theSHR (Figure 2B). To further evaluate theparticipation of ACE2 in the thrombo-genic process, the animals were treatedwith a specific ACE2 inhibitor (DX600)5 min before thrombus induction byFeCl3. Intravenous administration ofthis inhibitor produced an increase inthrombus weight by 46% in the SHRand 37% in the WKY (Figure 3A). Al-though ACE2 activity in thrombi fromthe SHR treated with DX600 decreased,the difference did not achieve statisticalsignificance (Figure 3C).

Figure 1. Increased thrombus formation in SHR is associated with lower ACE2 activity inthrombi. (A) FeCl2-induced thrombus weight in SHR and WKY; (B) ACE2 activity in thrombifrom SHR and WKY; (C) Representative Western blotting analysis of ACE2 levels. Data werenormalized by using β-actin. (D) Quantification of Western blotting data. *P < 0.05 versusWKY (unpaired Student t test, n = 8 to 10). A.U., arbitrary units.

Figure 2. ACE and ACE2 activity in thrombiof SHR and WKY rats. (A) ACE activity inthrombi of SHR and WKY. No significant dif-ferences were found between SHR andWKY. (B) Ratio between ACE2/ACE activityin thrombi of SHR and WKY. *P < 0.05 versusWKY (unpaired Student t test, n = 10 to 11).A.U., arbitrary units.



Effects of ACE2 Activation onAntithrombotic Activity

To address the effect of ACE2 activa-tion on thrombus formation, we usedtwo different models. The effect of XNT,a small-molecule ACE2 activator (9), onthrombus weight was evaluated first,using large vessels in rats. Second,platelet adhesion and thrombus forma-tion were viewed in real time usingmouse microvessels. XNT administered

through the jugular vein 5 min beforethrombus induction resulted in a de-crease in thrombus weight (34.7% in theSHR and 32.3% in the WKY) (Figure 3B).This effect was accompanied by an in-crease in ACE2 activity in the thrombi inthe SHR (Figure 3C). Moreover, treat-ment with XNT reduced platelet attach-ment to vessels and thrombus formationin mice, as observed under real-time in-travital microscopy (Figure 4A). Throm-bus area was reduced by 60%, whereastime for thrombus formation was pro-longed by 45% in XNT-treated mice (Fig-ure 4B, C).

DISCUSSIONThe most significant findings of the

present study are that ACE2 plays an im-

portant role in thrombogenic events andits activation by the small-molecule ACE2activator XNT has significant antithrom-bogenic effects. The role of ACE2 in thepathophysiology of cardiovascular dis-eases is currently under intense investiga-tion. The involvement of this enzyme incardiac contractile function, hyperten-sion, atherosclerosis and other cardiovas-cular diseases has recently been demon-strated (15–17). The present study offersevidence that ACE2 is involved in the he-mostatic process and exhibits a protectiveaction against thrombosis. We observedthat a decrease in ACE2 activity in thethrombi, but not in ACE2 protein, is asso-ciated with an increase in thrombus for-mation in the SHR. In keeping with thisfinding, the pharmacological activation of

Figure 3. Antithrombotic effect of XNT onrats. FeCl2-induced thrombus weight inWKY and SHR treated with (A) DX600 and(B) XNT. *P < 0.05 (unpaired Student t test).(C) ACE2 activity in thrombi of WKY andSHR treated with either XNT or DX600. *P <0.05 versus WKY rats, #P < 0.05 versus un-treated SHR (one-way ANOVA followed bythe Bonferroni posttest). Each column rep-resents mean ± SEM (n = 7 to 9).

Figure 4. Effect of XNT on thrombus development and time for thrombus formation. (A)Representative photomicrographs showing thrombi development in dorsal skin vessels innude mice recorded by use of intravital microscopy. The upper panel shows thrombi de-velopment in a control animal 1, 8 and 12 min after FeCl3 application. The lower panelshows thrombi development in an XNT-treated mouse 1, 8 and 12 min after FeCl3 applica-tion. Arrows indicate thrombi. (B) Ratio between thrombi area/vessel area. *P < 0.05 versusXNT treatment (two-way ANOVA followed by the Bonferroni posttest; n = 6 to 7). (C) Timefor thrombus formation after FeCl3 application. Each column represents mean ±SEM (n = 6to 7). *P < 0.05 versus vehicle (unpaired Student t test).


A N T I T H R O M B O T I C E F F E C T O F A C E 2

ACE2 using XNT produced a significantreduction in thrombus formation in theSHR to a level similar to that found in theWKY. XNT is a small-molecule ACE2 ac-tivator that was recently discoveredbased on the crystal structure of ACE2and a virtual screening strategy (9). Thiscompound enhances ACE2 activity,causes a reduction in arterial blood pres-sure and an impressive reversal of car-diac and renal fibrosis in SHR, and alsoprevents pulmonary hypertension (9,18).It is important to note that the increase inthrombus weight caused by DX600 ad-ministration in SHR (46%) and in WKY(37%) was not statistically different (P >0.05). One could argue that the inhibitionof ACE2 by DX600 should have greaterimpact on thrombus formation in WKYrats than in SHR, considering that ACE2activity is reduced in SHR. However, be-cause the balance between the two coun-terregulatory axes of the RAS is shiftedtoward the ACE/Ang II/AT1R branch inhypertensive rats, it is pertinent to specu-late that the responses to pharmacologicalmanipulations of the ACE2/Ang-(1-7)/Mas axis can produce distinct responses inhypertensive compared with normoten-sive animals.

The beneficial effects of ACE2 activa-tion are primarily due to the shifting ofthe balance of the RAS from the vaso-constrictive fibrotic axis to the vasodila-tor branch. This hypothesis is supportedby recent data (18). Although neitherAng II nor Ang-(1-7) levels in thethrombi were determined in the presentinvestigation, a large number of studieshave shown that Ang II is a prothrom-botic agent (19,20). Ang II is associatedwith an increase in the production andsecretion of plasminogen activator- inhibitor type 1 from endothelial andsmooth muscle cells and an augmenta-tion of tissue factor expression, therebyenhancing the activity of the coagulationsystem (19,20). Moreover, it is well es-tablished from previous studies thatplatelets express the AT1 receptor and itsactivation by Ang II potentiates plateletactivation and aggregation (21–23). Onthe other hand, Ang-(1-7) has been de-

scribed as an antithrombotic peptide(24). Kucharewicz et al. (24,25) haveshown that intravenous infusion ofAng-(1-7) produces a potent reductionin thrombus formation in renovascularhypertensive rats. Furthermore, theseauthors also found that the intrinsic an-tithrombotic effects of the antihyperten-sive drugs captopril (ACE inhibitor) andlosartan (AT1 receptor blocker) were at-tenuated by the selective Ang-(1-7) re-ceptor antagonist A-779 (24,25). More re-cently, it was demonstrated that theAng-(1-7) receptor Mas is present inplatelets and the interaction betweenAng-(1-7) and Mas on platelets promotesnitric oxide (NO) production, which is amajor antiplatelet agent (26). Moreover,the activation of endothelial Mas byAng-(1-7) has been reported to increaseNO and prostacyclin synthesis (27). Bothof these mediators are important anti -thrombotic agents in the hemostasis pro-cess (1,28). Because ACE2 can catalyzethe conversion of Ang II to Ang-(1-7), itis possible that the significant antithrom-botic effect observed with XNT treat-ment could be a result of a decrease inthe bioavailability of the prothromboticpeptide Ang II, followed by an increasein the formation of the antithromboticpeptide Ang-(1-7). Unfortunately, noMas antagonists were used in the pres-ent study. However, an experimentalmodel of pulmonary hypertensiondemonstrated that the protective effectsevoked by XNT were completely abol-ished by the coadministration of A-779(18). This important finding should betaken into consideration in future experiments.

The present study employed two dif-ferent in vivo models of thrombosis in-duced by FeCl3 to demonstrate the an-tithrombotic effects of XNT: thrombusformation in the abdominal vena cava ina rat model and in microveins of dorsalmouse skin. According to Kucharewiczet al. (25), an advantage of using theabove mentioned models is the abilityto separate antithrombotic and hypoten-sive effects of the compounds studied,because the thrombus forming inside

the veins is unlikely to be affected bychanges in arterial blood pressure (25).Because the relationship betweenchanges in arterial and venous bloodpressure is negligible, alterations in arte-rial blood pressure should not interferein thrombus formation in veins (25).Thus, it was possible to exclude the in-fluence of the hypotensive effect of XNT(9) from its antithrombotic action. More-over, the acute intravenous administra-tion of XNT at 10 mg/kg has been foundto cause a decrease in blood pressure inSHR and WKY rats; however, this effectis significantly lower in WKY comparedwith hypertensive rats (21.3 ±8.2 mmHgin WKY versus 71.0 ±9.0 mmHg in SHR)(9). Despite this difference, the antithrom-botic effect of XNT was similar in bothstrains (32.3% reduction in thrombusweight in the WKY versus 34.7% reduc-tion in thrombus weight in the SHR).These findings again suggest that the an-tithrombotic effect of XNT is indepen-dent from changes in blood pressure.Moreover, intravital microscopy allowedthe real-time visualization of the partici-pation of platelets in the thrombus for-mation process.

In summary, the results of this studydemonstrated that a decrease in ACE2activity in thrombi was associated withan increase in thrombus formation inSHR. Furthermore, the pharmacologicalactivation of ACE2 attenuated plateletattachment to vessels and thrombus for-mation. These results clearly suggestthat XNT could be a potential lead com-pound for the treatment of thrombo-genic diseases.

ACKNOWLEDGMENTSThis work was supported by the Na-

tional Institutes of Health Grant HL56912.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-sults and discussion reported in thispaper.



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ACE2 Activation Promotes Antithrombotic Activity