Renal Damage after Myocardial Infarction Is Prevented by Renin-Angiotensin-Aldosterone-System Intervention
Willemijn A.K.M. Windt*,
Wouter B.A. Eijkelkamp*,
Robert H. Henning*,
Alex C.A. Kluppel*,
Pieter A. de Graeff*,
Hans L. Hillege,
Stefan Schäfer,
Dick de Zeeuw* and
Richard P.E. van Dokkum*
* Department of Clinical Pharmacology and Department of Cardiology, Thorax Center, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; and Sanofi-Aventis Pharma Deutschland, Frankfurt am Main, Germany
Address correspondence to: Dr. Richard P.E. van Dokkum, Department of Clinical Pharmacology, Groningen Institute for Drug Exploration, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, NL-9713 AV, Groningen, The Netherlands. Phone: +31-50-363-2840; Fax: +31-50-363-2812; E-mail: r.p.e.van.dokkum{at}med.umcg.nl
Received for publication March 7, 2006.
Accepted for publication August 9, 2006.
Recently, it was shown that myocardial infarction aggravatespreexistent mild renal damage that is elicited by unilateralnephrectomy in rats. The mechanism behind this cardiorenal interactionlikely involves the renin-angiotensin-aldosterone-system and/orvasoactive peptides that are metabolized by neutral endopeptidase(NEP). The renoprotective effect of angiotensin-converting enzymeinhibition (ACEi) as well as combined ACE/NEP inhibition witha vasopeptidase inhibitor (VPI) was investigated in the samemodel to clarify the underlying mechanism. At week 17 aftersequential induction of unilateral nephrectomy and myocardialinfarction, treatment with lisinopril (ACEi), AVE7688 (VPI),or vehicle was initiated for 6 wk. Proteinuria and systolicBP (SBP) were evaluated weekly. Renal damage was assessed primarilyby proteinuria, interstitial -smooth muscle actin (-SMA) staining,and the incidence of focal glomerulosclerosis (FGS). At startof treatment, proteinuria had increased progressively to 167± 20 mg/d in the entire cohort (n = 42). Both ACEi andVPI provided a similar reduction in proteinuria, -SMA, and FGScompared with vehicle at week 23 (proteinuria 76 ± 6versus 77 ± 4%; -SMA 60 ± 6 versus 77 ±3%; FGS 52 ± 14 versus 61 ± 10%). Similar reductionsin systolic BP were observed in both ACEi- and VPI-treated groups(33 ± 3 and 37 ± 2%, respectively). Compared withACEi, VPI-treated rats displayed a significantly larger reductionof plasma (41 ± 5 versus 61 ± 4%) and renal (53± 6 versus 74 ± 4%) ACE activity. It is concludedthat both ACEi and VPI intervention prevent renal damage ina rat model of cardiorenal interaction. VPI treatment seemedto provide no additional renoprotection compared with sole ACEiafter 6 wk of treatment in this model, despite a more pronouncedACE-inhibiting effect of VPI.
Recent studies have disclosed a complex relationship betweencardiovascular and renal disease. Impaired renal function isdetrimental for the heart (renocardiac interaction) both inclinical (1–8) and in experimental (9,10) settings. Impairedcardiac function is detrimental to the kidney (cardiorenal interaction),which is less well characterized and only in experimental settings.Recently, we described enhanced progressive renal damage ina model of cardiorenal interaction that was elicited by myocardialinfarction (MI) in unilaterally nephrectomized rats (11). Themechanism of this cardiorenal interaction is unclear, but candidatemechanisms are hormonal systems such as the renin-angiotensin-aldosterone-system(RAAS) and the natriuretic peptide system (11). Both angiotensin-convertingenzyme inhibition (ACEi) and neutral endopeptidase (NEP) inhibition(although controversial) have been shown to be protective inprogressive function loss of either the kidney or the heart(12–15). The combination of ACE and NEP inhibition isclaimed to be more effective in this regard (16–20). Accordingly,the aim of our study was to investigate involvement of the RAASand/or the natriuretic peptide system in the pathophysiologyof progressive renal damage after MI. We investigated the renoprotectiveeffects of ACEi with lisinopril versus combined ACE/NEP inhibitionwith the vasopeptidase inhibitor AVE7688 (21–23).
Experimental Protocol
Male Wistar rats (320 to 350 g; n = 75) were housed under standardconditions with free access to food and drinking water. Ratsreceived a standard chow diet that contained 19% protein (diet#1324; Altromin, Lage, Germany). Animal experiments were approvedby the institutional animal ethics committee.
At week –2, all 75 rats underwent right unilateral nephrectomyunder anesthesia with 2.0% isoflurane in N2O/O2 (2:1) as describedpreviously (11). At week 0, rats were intubated, ventilated(AIV, Hoek Loos, The Netherlands), and anesthetized using 2.0%isoflurane in O2. MI was induced by ligation of the left anteriordescending coronary artery as described previously (24). Ofthese 75 rats, 30 died within 24 h after MI surgery and wereexcluded from the study. Seventeen weeks after MI, rats werestratified for proteinuria as a measure for renal damage andassigned to one of the following treatment groups: (1) vehicle(n = 12), (2) lisinopril 5 mg/kg per d (ACEi; n = 16), and (3)AVE7688 21 mg/kg per d (VPI; n = 14). A 6-wk treatment periodwas initiated up to week 23, when the rats were killed. Lisinopril(Merck, Sharp & Dohme, Haarlem, The Netherlands) was administeredthrough the drinking water in a concentration of 75 mg/L, providinga dosage of 5 mg/kg per d. AVE7688 (Sanofi-Aventis Pharma Deutschland,Frankfurt am Main, Germany) was mixed through the food at aconcentration of 450 ppm, resulting in a dosage of 21 mg/kgper d. In a pilot experiment, these dosages resulted in comparableplasma ACEi. At the end of the experiment, after measurementof functional cardiac parameters under 2.5% isoflurane anesthesia,laparotomy was performed and rats were exsanguinated by takingblood samples from the abdominal aorta for plasma measurements.The remaining kidney was flushed with saline, and the heartand the kidney were removed and weighed.
Functional Parameters BP and Urinary Measurements.
Systolic BP (SBP) was measured using tail-cuff plethysmography(IITC Life Science, Woodland Hills, CA) in trained awake, restrainedrats. SBP was measured at baseline and regularly thereafteruntil the end of study. Measurements of water and food intakeas well as 24-h urine collections for determination of urinarytotal protein excretion and urine production were performedweekly by placing the rats in metabolic cages.
Urine, Plasma, and Tissue Measurements.
Urinary total protein was analyzed using end-point measurementwith TCA precipitation (Nephelometer Analyzer II; Dade Behring,Marburg, Germany). As a representation of renal function, creatinineclearance was calculated from urinary and plasma creatininelevels at baseline, at stratification, and at the end of theexperiment. Creatinine was determined using standard kits (RocheDiagnostics, Basel, Switzerland) on a Hitachi 912 E analyzer(Hitachi, Mountain View, CA). Plasma and renal ACE activitywere measured to evaluate the effect of treatment on ACEi usingthe conversion of hippuryl-His-Leu (Sigma, St. Louis, MO) byACE to free His-Leu (25). Plasma renin activity was measuredto investigate the activity of the RAAS by a RIA (Adaltis ItalySPa, Bologna, Italy), and pro-atrial natriuretic peptide (pro-ANP)was measured to investigate NEP inhibition using a sandwichenzyme immunoassay (Biomedica, Vienna, Austria).
Cardiac Function at the End of the Study
Under 2.5% isoflurane in O2 anesthesia, cardiac performancewas measured with a pressure transducer catheter that was insertedthrough the right carotid artery (Micro-Tip 3French; MillarInstruments Inc., Houston, TX), connected to a personal computerthat was equipped with an analog-to-digital converter and appropriatesoftware (Millar Instruments). After a 3-min period of stabilization,left ventricular end diastolic pressure (LVEDP), left ventricularend systolic pressure (LVESP), and heart rate were recorded.Thereafter, the catheter was withdrawn into the aortic rootto measure central SBP (SBPcentral). As a parameter of globalmyocardial contractility and relaxation, we determined the maximalrates of increase and decrease in left ventricular pressure(LVP) (systolic +dP/dtmax and diastolic –dP/dtmax), whichwere normalized to left ventricular pressure change (i.e., LVESP– LVEDP) for individual rats.
Histology Kidney.
Kidneys were fixed by immersion for 48 h in a 4% buffered formaldehydesolution (Klinipath, Duiven, The Netherlands) after longitudinalbisection and subsequently embedded in paraffin according tostandard procedures. An examiner who was blinded for the groupsevaluated all sections.
Mesangial Matrix Expansion, Focal Glomerulosclerosis, and Interstitial Fibrosis.
Sections of 3 µm were stained with periodic acid Schiff.The degree of mesangial matrix expansion (MME) and focal glomerulosclerosis(FGS) were assessed in 50 glomeruli by scoring semiquantitativelyon a scale of 0 to 4 (26). FGS was scored positive when MMEand adhesion to Bowman’s capsule were present in the samequadrant. When one quadrant of the glomerulus was affected,a score of 1+ was assigned, two quadrants was scored as 2+,three quadrants as 3+, and four quadrants as 4+. Overall MMEand FGS score is expressed in arbitrary units (AU) with a maximumof 200. Interstitial fibrosis (IF) was defined as expansionof the interstitial space, with or without the presence of atrophiedand dilated tubules and thickened tubular basement membranes.The degree of IF was assessed in 30 interstitial fields at x20magnification by scoring semiquantitatively on a scale of 0to 5 as follows: 0, no IF; 1, 1 to 10%; 2, 10 to 25%; 3, 25to 50%; 4, 50 to 75%; and 5, 75 to 100%. The score is givenas AU with a maximum of 150.
Interstitial and Glomerular -Smooth Muscle Actin and Glomerular Surface Area. -Smooth muscle actin (-SMA) was determined as a profibroticmarker and detected in paraffin-embedded sections by means ofa mouse monoclonal -SMA antibody (Sigma Chemical). First, theantibody was incubated for 60 min, and its binding was detectedby sequential incubations with peroxidase-labeled rabbit anti-mouseand peroxidase-labeled goat anti-rabbit antibody (both fromDakopatts, DAKO, Glostrup, Denmark) for 30 min. The expressionof interstitial -SMA was measured by computerized morphometry.Therefore, 40 fields were scored at x20 magnification in thecortical region; glomeruli and vessels were excluded from measurementalong Bowman’s capsule and the vessel wall. Glomerularsurface area, as a measure for glomerular hypertrophy, was measuredusing this procedure as well. For the expression of glomerular-SMA, 40 glomeruli were scored at x20 magnification. Total stainingwas expressed as percentage of total area surface.
Interstitial Macrophage Number.
The number of interstitial macrophages was determined as anindication of the degree of inflammation. Therefore, a mousemonoclonal anti-rat monocyte and macrophage IgG1 (ED-1; Serotec,Oxford, England) was used. First, the antibody was incubatedfor 60 min, and its binding was detected by sequential incubationswith peroxidase-labeled rabbit anti-mouse and peroxidase-labeledgoat anti-rabbit antibody (both from Dakopatts, DAKO) for 30min. The expression of interstitial ED-1—positive cellsper field was measured by computerized morphometry. Therefore,40 fields were scored at x20 magnification in the cortical region;glomeruli were excluded from measurement along Bowman’scapsule. The average score was calculated per cortical section.
Glomerular Desmin.
Desmin, a marker for glomerular visceral epithelial cell damage,was detected using a mouse mAb (clone DE-R-11; Novocastra LaboratoriesLtd, Newcastle, UK). For glomerular desmin staining, 30 glomeruliwere scored semiquantitatively, by estimating the percentageof desmin-positive glomerular visceral epithelial cells (injuredpodocytes) in the outer cell layer of the glomerular tuft from0 to 5 as follows: 0, no staining; 1, 1 to 10%; 2, 10 to 25%;3, 25 to 50%; 4, 50 to 75%; and 5, 75 to 100% staining. Desminstaining is presented in AU with a maximum of 150.
Heart. Infarct Size.
The heart was arrested in diastole in a cold 1-M KCl solutionand weighed. The atria were dissected from the ventricles, andthe right free wall was separated from the left ventricle andweighed. Two left ventricular midsagittal slices (of approximately2 mm) were fixed in 4% buffered formaldehyde solution, embeddedin paraffin, cut into 5-µm slices, and stained with 0.1%Sirius Red F3B (Klinipath, Duiven, The Netherlands) and 0.1%Fast Green FCF (Klinipath). Endo- and epicardial circumferenceof the left ventricle and of scar tissue was determined by meansof a computerized planimeter (Image-Pro plus; Media CyberneticsInc., Silver Spring, MD). MI size was expressed as the meanof the inner and outer percentage of scar tissue to the innerand outer total circumference of the left ventricle. All sectionswere evaluated by an examiner who was blinded for the groups.
Statistical Analyses
All data are presented as mean ± SEM. In general, differencesbetween the groups were compared using a one-way ANOVA for parametersmeasured at one time point and an analysis of covariance forparameters with two repeated measurements before and after thetreatment period. A general linear model for repeated measureswas used to compare the change in proteinuria and change inSBP curves during the treatment phase (weeks 18 through 23).A Bonferroni post hoc test was used to identify the differencesbetween groups. A paired samples t test was used to comparea parameter before and after treatment in one group. In alltests, P < 0.05 was considered statistically significant.
Overall Condition
In the vehicle- and ACEi-treated groups, body weight remainedstable during the treatment period, even as food intake andurine production (Table 1). In the VPI-treated group, body weightsignificantly decreased during this period, whereas food intakeremained stable and water intake increased. Differences in urineproduction before and after treatment were not observed betweengroups. Water intake was significantly higher in the ACEi- andthe VPI-treated groups compared with vehicle.
Effects of Treatment Regimens on Plasma and Renal ACE Activity, Levels of Renin, and Pro-ANP
At the end of the treatment period, plasma ACE activity wassignificantly lower (41 ± 5%) in the ACEi-treated groupand in the VPI-treated group (61 ± 4%) compared withvehicle (Table 2). Renal ACE activity was significantly lowerin the ACEi-treated group (53 ± 6%) and in the VPI-treatedgroup (74 ± 4%) compared with vehicle (Table 2). Plasmaand renal ACE activity was significantly more inhibited in theVPI- compared with the ACEi-treated group. Plasma renin activitywas significantly and equally higher in both the ACEi- and theVPI-treated groups (Table 2) compared with vehicle. Pro-ANPlevels were significantly higher in the VPI- compared with theACEi-treated group (Table 2).
Table 2. Treatment effects at the end of the study
Cardiovascular Characteristics
Tail-cuff SBP remained stable from a baseline level of 134 ±2 to 137 ± 3 mmHg at stratification. At the end of thetreatment period, the groups that were treated with ACEi andVPI showed a significant reduction in SBP compared with stratification(33 ± 3 and 37 ± 2%, respectively), whereas SBPremained stable in the vehicle-treated group (Figure 1A). TheBP-lowering effect was comparable in the ACEi- and VPI-treatedgroups. Intra-arterially measured SBP (at the end of the study)showed a comparable difference between vehicle and both treatmentgroups (20 ± 2 and 26 ± 3% lower in ACEi and VPI,respectively; Table 3).
Figure 1. Effect of treatment with angiotensin-converting enzyme inhibitor (ACEi) and vasopeptidase inhibitor (VPI) on systolic BP (SBP; A) and proteinuria (B), given as change in SBP and change in proteinuria from stratification (week 17). *P < 0.001 versus vehicle (VEH).
Table 3. Cardiac parameters at the end of the studya
MI size was comparable in all groups (Table 3). Total wet heartweight was significantly lower in the ACEi- and VPI-treatedgroups compared with the vehicle-treated group (Table 3).
LVESP and LVEDP were significantly lowered in the ACEi- andVPI-treated groups compared with the vehicle-treated group (Table 3)and therefore were not different between ACEi and VPI. The maximalrate of left ventricular isovolumetric pressure development(+dP/dtmax) was significantly higher in the ACEi- compared withthe vehicle-treated group. A similar trend, although not statisticallysignificant, was observed in the VPI-treated group (Table 3).The isovolumetric pressure decay (–dP/dtmax) was not affectedby therapy.
Renal Characteristics
Daily urinary total protein excretion increased in the entirecohort from a baseline level of 16 ± 1 to 167 ±20 mg/d at stratification with comparable levels in all groups(Table 1). After a subsequent treatment period of 6 wk, proteinuriawas significantly lower in the ACEi-treated group (76 ±6%) and in the VPI-treated group (77 ± 4%) compared withthe vehicle-treated group, in which proteinuria steadily increasedto 224 ± 45 mg/24 h (Table 1). There was no significantdifference in antiproteinuric effect between both treatments(Figure 1B). At the end of the treatment period, proteinuriawas reduced by ACEi to 105 ± 22 mg/d compared with stratificationand by VPI to 110 ± 28 mg/d.
When treatment was started, creatinine clearance was significantlyreduced from a mean level of 10.6 ± 0.5 ml/min per kgat baseline to 4.5 ± 0.2 ml/min per kg for the entirecohort, corresponding to serum creatinine level of 42 ±1 µmol/L. Although no significant difference in creatinineclearance was observed at stratification between the ACEi- andVPI-treated groups, there was a difference between the ACEi-and vehicle-treated groups (Table 1). Corrected for the levelsat stratification, no difference in treatment effect on creatinineclearance was observed.
At the end of the study, no significant differences were presentin the wet weight of the remaining left kidney. Glomerular damagewas investigated by measurement of glomerular surface area,FGS, MME, glomerular -SMA staining, and glomerular desmin staining(Figure 2). Glomerular surface area was significantly smallerin ACEi- compared with the vehicle-treated group. VPI treatmentdid not affect glomerular surface area. FGS was significantlylower in ACEi- compared with the vehicle-treated group, anda trend toward a lower level was observed for the VPI-treatedgroup (P = 0.11). Glomerular -SMA staining was significantlylower after treatment with both ACEi and VPI compared with thevehicle-treated group. No significant differences in MME wereobserved after treatment with ACEi and VPI compared with vehicle.The level of desmin staining, as a measure for podocyte damage,was significantly lower after VPI treatment compared with bothACEi and vehicle. Interstitial damage was investigated by themeasurement of IF, interstitial -SMA staining, and interstitialED-1 staining (Figure 2). A trend toward lower levels of IFwas observed after treatment with ACEi (P = 0.2) and VPI (P= 0.2) compared with vehicle. A significant lower interstitial-SMA staining was observed in both the ACEi- and the VPI-treatedgroups compared with the vehicle-treated group. A similar patternwas seen for the number of ED-1–positive cells.
This study confirms our previous finding that MI induces enhancedprogressive renal damage in unilateral nephrectomized rats (11).This detrimental cardiorenal interaction can be attenuated bytreatment with either an ACE inhibitor or a VPI, with substantialbeneficial effects on kidney and heart. It is interesting thatthe addition of NEP inhibition on top of ACEi (as offered bythe VPI) was devoid of extra protection.
Several mechanisms have been hypothesized, including hemodynamicalterations, involvement of the RAAS and sympathetic nervoussystem, endothelial dysfunction, and inflammation (27), whichalso are thought to interact with each other. First, as faras the role of hemodynamics in explaining the observed cardiorenalinteraction is concerned, reduced cardiac output after MI maylead to reduced renal perfusion, which in turn could lead tocompensatory RAAS activation. This RAAS activation in turn canbe detrimental to both heart and kidney. An elevated angiotensinII level is known to interact with cardiac function, leadingto progressive cardiac function loss (28), and elevated angiotensinII levels may lead to progressive renal damage (29). It is interestingthat RAAS intervention with either ACEi or angiotensin II receptorblocker can protect both heart and kidney (30,31). In our cardiorenalinteraction model, ACEi therapy was effective in the preventionof enhanced renal damage that was caused by MI, because thelevel of proteinuria, interstitial and glomerular -smooth muscleactin staining, glomerular surface area, and FGS incidence weresignificantly lower compared with those in the vehicle group.The level of renal inflammation (ED-1 staining) seemed to besignificantly lower in the ACEi-treated group. It is interestingthat cardiac contractility (+dP/dtmax) and LVEDP showed a morefavorable outcome in the ACEi- compared with the vehicle-treatedgroup at the end of the study. In view of these findings, itis likely that the RAAS is of significant importance in thiscardiorenal interaction model. It should be taken into account,however, that RAAS inhibition is invariably associated withBP reduction. Therefore, lowering BP by means of a calcium channelblocker would be an interesting strategy to allow further fora distinction between BP lowering per se and blockade of theRAAS.
Regarding VPI treatment, it has been postulated that natriureticpeptides act on both the heart and the kidneys by vasodilation,natriuresis, diuresis, decreased cell growth, inhibition ofthe sympathetic nervous system, and inhibition of the RAAS (32).In our previous study, we found a trend toward a decrease innatriuretic peptide levels in the group with combined cardiacand renal damage, whereas in the group with only cardiac damage,higher levels were observed (11). From this observation, wehypothesized a role for natriuretic peptides in the deterioratingeffect of the cardiorenal interaction. However, no additionalprotective effect of VPI over ACEi on renal damage as measuredas FGS, proteinuria, and interstitial and glomerular -smoothmuscle actin staining was observed in our study. Although VPIwas more effective than ACEi in prevention of podocyte damage,this did not result in the expected augmented prevention ofincreased proteinuria or FGS (33). Overall, this leaves onlya little evidence for a discernible beneficial effect of anincreased level of natriuretic peptides beyond concurrent RAASinhibition and associated BP reduction in this cardiorenal modelwith short-term pharmacologic intervention. Our study, however,does not exclude VPI still to have an important clinical contributionin both "renal" and "cardiac" patients, because VPI have provedto be effective in more isolated renal and cardiovascular disease(18–20,34).
Some caution is needed when interpreting the efficacy of ACEiversus VPI treatment. First, an important issue relates to findingequipotent dosages with respect to effects on BP and plasmaACE activity. Although we performed a pilot experiment to determinedosage levels that yield a comparably reduced level of plasmaACE activity with ACEi and VPI treatment in healthy rats, ACEactivity was not reduced similarly by both treatment regimensin our study, although effects on BP were comparable. Besidesthis, no full dose-response relationships were established inour study. However, this would not change substantially theinterpretation of the data, because the selected VPI dosageresulted in a larger reduction in ACE activity. The effectson proteinuria, -SMA, and FGS were comparable. This might indicatea dissociation between the level of ACEi and antiproteinuriceffect of these drugs. Second, despite the substantial increasein pro-ANP levels in the VPI- compared with the ACEi-treatedgroup, the NEP inhibiting component of the VPI may have insufficientlyincreased the levels of natriuretic peptides at the selecteddosage to provide cardiorenal protection. Third, a VPI treatmentperiod that is longer than 6 wk may be required for the beneficialcardiorenal effects of ANP to develop. Taal et al. (18) showedthat the VPI omapatrilat displayed favorable effects comparedwith the ACEi enalapril at a treatment duration of 32 wk infive-sixths nephrectomized rats, despite comparable efficacyin the short term. Further long-term studies are needed to clarifythis issue.
Our results demonstrate the efficacy of RAAS-inhibiting therapyto prevent the enhanced progression of renal damage after MIin a model of mildly compromised renal dysfunction. This interventionis of potential clinical relevance, because it breaks a viciouscircle: MI leading to cardiac dysfunction in turn leading torenal dysfunction triggering further cardiac dysfunction. This,however, will require changes in prescribing behavior of doctorswho treat patients after MI. Patients are not regularly prescribedRAAS intervention after MI (35), whereas "cardiac" patientswith compromised renal function are being approached with utmostcare as far as RAAS intervention is concerned (36). This isin favor of the recent view that these drugs reduce long-termcardiovascular morbidity and mortality, apart from their directeffects on BP and proteinuria. Our study further indicates thatRAAS intervention after MI may provide not only cardiac protectionbut also renal protection.
Acknowledgments
This study was financially supported by Sanofi Aventis PharmaDeutschland.
We acknowledge J.J. Duker, Dr. M. Gerl, M. Goris, Dr. H. vanGoor, B.G. Haandrikman, F. Hut, B. Meijeringh, Dr. R. Schoemaker,J.W.J.T van der Wal, and C. Wisselink for valuable advice andtechnical assistance. Lisinopril was a kind gift from Merck,Sharp & Dohme (Haarlem, The Netherlands). AVE7688 was akind gift from Sanofi Aventis Pharma Deutschland.
Parts of this study were presented at the American Society ofNephrology Renal Week; November 8 through 13, 2005; Philadelphia,PA; and have been published in abstract form (J Am Soc Nephrol16: 215A, 2005).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
W.A.K.M.W. and W.B.A.E. contributed equally to this work.
S.S.’s current affiliation is Bayer Healthcare AG, Wuppertal,Germany.
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Top
Abstract
Introduction
-smooth muscle actin (
, VEH; 
, VPI. *P < 0.05 versus VEH; #P < 0.05 versus VEH and ACEi. Magnifications: x40 in A and D; x20 in B, C, and E.