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Published ahead of print on December 21, 2005
J Am Soc Nephrol 17: 433-441, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2005080883
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Hemodynamics and Vascular Regulation

Antiatherogenic Effects of Angiotensin Receptor Antagonism in Mild Renal Dysfunction

Eisuke Suganuma*, Yiqin Zuo*, Nobuhiko Ayabe*, Ji Ma*, Vladimir R. Babaev{dagger}, MacRae F. Linton{dagger}, Sergio Fazio{dagger}, Iekuni Ichikawa*, Agnes B. Fogo{dagger},{ddagger} and Valentina Kon*

Departments of * Pediatrics, {dagger} Medicine, and {ddagger} Pathology, Vanderbilt University Medical Center, Nashville, Tennessee

Address correspondence to: Dr. Valentina Kon, Vanderbilt University Medical Center, 1161 21st Avenue South, C-4204 Medical Center North, Nashville, TN 37232-2584. Phone: 615-322-7416; Fax: 615-322-7929; E-mail: valentina.kon{at}vanderbilt.edu

Received for publication August 26, 2005. Accepted for publication November 1, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (Ang II) increases atherosclerotic cardiovascular disease. Renal damage that is characterized by activation of Ang II markedly potentiates the risk for atherosclerosis, even in the setting of subtle renal impairment. Therefore, whether antagonism of Ang II actions can modify atherosclerosis in a model of mild renal impairment was examined. Apolipoprotein E–deficient spontaneously hyperlipidemic mice underwent uninephrectomy (UNx) or sham operation (sham) followed by treatment with Ang II receptor antagonist losartan or hydralazine for 12 wk. While UNx did not increase the serum creatinine levels, BP and lipids were higher in UNx mice than in age-matched sham controls with intact kidneys. UNx caused a dramatic increase in the extent and the number of atherosclerotic lesions together with greater macrophage-positive area and more disruption in the elastin component of the extracellular matrix versus sham. Ang II antagonism dramatically decreased the UNx-induced acceleration in atherosclerosis in association with decreased macrophage content, linked to decreased macrophage migration in vitro with losartan but not with hydralazine. Aortae of mice treated with Ang II antagonism had fewer elastin breaks together with less immunostaining for the powerful elastolytic enzyme cathepsin S. None of these benefits was observed in the hydralazine-treated mice despite equivalent reduction in BP. These findings support an important role for endogenous Ang II in accelerated atherosclerosis in renal dysfunction and offer a therapeutic intervention with particular benefit in this setting through mechanisms that include reduced vascular macrophage infiltration and preservation of the elastin component of extracellular matrix.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Patients with chronic kidney disease (CKD) are now recognized to compose the "highest risk group" for cardiovascular disease (CVD) events, even greater than individuals with diabetes (1). Importantly, increased cardiovascular events prevail at every stage of CKD, even in the setting of very subtle renal impairment (1–6). These observations are significant not only because of the well-documented increase in the incidence and the prevalence of end-stage CKD but also because the number of patients with early CKD exceeds the number of patients with end-stage CKD by a factor of 30 to 60 (1). In this connection, a recent study of the natural history of patients with early CKD found that while approximately 3.1% of patients with early CKD progressed to dialysis or transplantation, 24.9% died over the same time period, many apparently from CVD (3). Thus, the magnitude of the CVD problem is unambiguous; it remains unclear which risk factors impart the heightened risk. Recognition of risks is important because implementation of risk factor–reducing programs and better therapeutic interventions has already lessened the overall morbidity and mortality in the general population. By contrast, no such trend has occurred in patients with CKD (7). Indeed, patients with CKD undergo fewer diagnostic analyses and receive fewer therapeutic interventions, including fewer lipid-lowering or antiplatelet agents, fewer beta blockers, and fewer angiotensin blockers for treatment of coronary artery disease (7). This is especially noteworthy because angiotensin II (Ang II) antagonism is the premiere medical therapy for progressive renal dysfunction for which heightened Ang II is thought to promote vasoconstriction, proteinuria, cellular adhesion, proliferation, and hypertrophy as well as dysregulation of extracellular matrix (ECM) (8–10). Many of these same processes that contribute to atherogenesis have been linked to Ang II actions (11,12). It is of interest, therefore, that epidemiologic studies of people without renal disease find fewer cardiovascular events and increased survival of individuals on Ang II antagonists compared with other antihypertensives (13–15). Because patients with CKD are often excluded from epidemiologic studies of potential therapies, little is known about the potential role of Ang II or the effects of Ang II antagonists in CKD-accelerated atherosclerosis.

Limitations in availability of patient data have been exacerbated by the absence of suitable animal models to study the impact of renal dysfunction on mechanisms of development and progression of atherosclerotic vasculopathy. However, genetically engineered apolipoprotein E–deficient (apoE–/–) mice, which have delayed clearance of lipoproteins, develop hyperlipidemia and spontaneous atherosclerosis that recapitulates many features of human atherosclerosis (16). Several recent reports have documented that reduction of renal parenchyma in apoE–/– mice accelerates atherosclerosis (17–20). As in intact mice, the atherosclerotic lesions in mice with reduced renal parenchyma contain macrophages; collagen, markers of inflammation, proliferation, and endothelial cell activation; oxidative stress; upregulation in intercellular adhesion molecules; and calcification. Thus, although there is considerable potential for renal dysfunction–activated Ang II to modulate atherosclerosis, no published studies have specifically investigated this possible mechanism. These studies were designed to evaluate the role of Ang II in renal damage–induced acceleration of atherosclerosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Experimental Design
Female apoE–/– mice on C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained on a normal mouse chow diet (RP5015, PMI Feeds, St. Louis, MO). Animal care and procedures were carried out in accordance with National Institutes of Health and Vanderbilt University animal care facility guidelines. At 8 wk of age, the mice underwent uninephrectomy (UNx) or sham operation (sham) performed under sodium pentobarbital anesthesia (50 mg/kg body wt, intraperitoneally). Four weeks after UNx or sham, the mice were divided into four groups: UNx with no treatment (UNx, n = 12); sham-operated with no treatment (sham; n = 4); UNx plus Ang II receptor antagonist (losartan, 100 mg/L drinking water [UNx+losartan]; n = 11); and UNx plus treatment with a nonspecific antihypertensive drug (hydralazine, 60 mg/L drinking water [UNx+hydralazine]; n = 5). All mice were killed 12 wk later. For in vitro macrophage migration studies, 10 additional apoE–/– mice with intact kidneys were used to harvest the macrophages (see below).

Systemic Parameters
Systemic BP was determined using Muramachi Systems (Model MK-2000; Muramachi Kikai, Osaka, Japan) automated tail-cuff system in conscious mice that were acclimated to the procedure. Determinations were obtained every 1 to 2 wk and before the mice were killed. Each measurement was repeated three times for every animal to yield a mean value. Body weight was obtained at the beginning and at the end of the experiment. Serum and urine creatinine, cholesterol, and triglyceride levels were determined at the end of the experiment as described previously (21–23). Urine was obtained from 24-h metabolic cage samples before killing in six sham, eight UNx, four UNx+losartan, and three UNx+hydralazine mice, and volume was measured. The creatinine was analyzed using the Roche Modulars P800.

Quantification of Atherosclerotic Lesions
Mice were killed under phenobarbital anesthesia (50 mg/kg body wt intraperitoneally), and perfused with PBS through the left ventricle. Heart, together with proximal aorta, was embedded in OCT and snap-frozen in liquid nitrogen. Cryosections, 10 µm thick, were cut from the proximal aorta beginning at the end of the aortic sinus with modifications specific for computer analysis (21–23). Cryosections were stained with Oil-Red-O and counterstained with hematoxylin (Sigma, St. Louis, MO). Quantitative analysis of lesions was performed using Imaging System KS300 (Release 2.0; Kontron Elektronik GmbH, Poway, CA) on at least 15 sections from each animal. The entire aorta, from the aortic valves to the iliac bifurcation, was dissected, and the en face preparations opened longitudinally, pinned flat, and stained with Sudan IV. The atherosclerotic lesions were compared by using computerized analysis with lesions expressed as percentage of total vascular surface (21–23). Numbers of Sudan IV–positive plaques in en face aorta were assessed by counting. The operator was blinded to the group assignment.

Immunohistochemistry
Serial 5-µm-thick cryosections from proximal aortae were fixed in acetone for (MOMA-2) or 4% paraformaldehyde. For immunohistochemistry, monoclonal rat antibody to mouse macrophages (MOMA-2, Serotec, Raleigh, NC) was used to detect macrophage infiltration, followed by incubation with biotinylated rat antibodies to rat IgG (PharMingen, San Diego, CA). The sections were treated with avidin-biotin complex labeled with alkaline phosphatase (Vector Laboratories, Burlingame, CA). Macrophage infiltration was visualized with Fast Red/Naphthol AS-TM substrate (Sigma). For determination of the macrophage infiltration in the lesions, the area that was stained with MOMA-2 was measured using Imaging System KS-300 and calculated as the ratio of macrophage-stained to Oil-Red-O–stained areas as described previously (23). Sections that were stained with the polyclonal cathepsin S antibody (1:100 dilution; Calbiochem, San Diego, CA) were incubated overnight and subsequently incubated with secondary antibodies (Vector) followed by incubation with ABC-AP complex. Cathepsin S was visualized using Alkaline Phosphatase Substrate Kit I and levamisole (24). In each experiment, negative controls without the primary antibody were included and showed no staining.

Histology
Verhoeff-van Gieson staining for elastin was performed in samples along the entire aorta as described previously (21). Briefly, starting from the proximal-most part, the aortae were divided into five sections that were embedded in paraffin. Each of the five parts was cut into 4-µm sections and stained for elastin by the Verhoeff-van Gieson method (Elastin stain kit; Newcomer Supply, Middleton, WI). Elastin breaks were defined by interruption in the elastin fiber together with the reappearance of elastic fiber under high-power field microscopy as described previously (24). Five sections were assessed such that at least 25 sections that represented five different areas along the aorta were included in the determination in each animal. The number of breaks was related to the thickness of the medial area and expressed as number of elastin breaks per aortic section. Medial area, defined between internal elastic lamina and external elastic lamina, was measured with an image analysis system KS-300 (Kontron Elektronik GmbH).

Macrophage Migration
Macrophage migrating activity was assessed in peritoneal macrophages that were collected from additional apoE–/– mice with intact kidneys (n = 10). After peritoneal injection of 3% thioglycolate, the cells were harvested by peritoneal lavage as described previously (21,25). The macrophages were pooled and then exposed for 90 min to Ang II (10–6 or 10–7M) alone or Ang II together with losartan (10–5 M) or Ang II together with hydralazine (20 µM) (26,27). The ex vivo studies were performed in a 96-well microchemotaxis chamber in which the upper compartment is separated from the lower compartment by a single uncoated polycarbonate filter (Neuroprobe, Gaithersburg, MD). Monocyte chemoattractant protein-1 (0.1 µg/ml; Preprotech, Rocky Hill, NJ) was added to the lower compartment and incubated at 37°C for 1 h. Filters then were fixed in methanol and stained with 1% crystal violet. Migrated cells that adhered to the lower surface of the membrane were counted manually under the microscope. Five separate experiments were performed. Quadruplicate wells were used for each experimental condition, and more than three fields (x40) were counted for each well.

Statistical Analyses
Results are expressed as mean ± SEM. Statistical difference was assessed by a single-factor variance (ANOVA) followed by unpaired t test as appropriate. Nonparametric data were compared by Mann-Whitney U test. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systemic Parameters
Table 1 shows the whole-body parameters. There were no differences in body weight among the groups at any time. Before treatment at 12 wk of age, there were no differences in systolic BP among the groups. When the mice were killed at 24 wk of age, only the untreated UNx mice had elevated BP. Importantly, although BP in losartan-treated mice was lower than in UNx mice, it was indistinguishable from the BP levels in hydralazine-treated animals. Creatinine at the time of killing was not different among the groups. Creatinine clearance measurements at the end of the study also showed no difference in renal function among the groups (sham 9.2 ± 1.1 ml/min per g body wt; UNx 10.2 ± 1.0 ml/min per g body wt; UNx+losartan ml/min per g body wt: 13.3 ± 1.3 ml/min per g body wt; UNx+hydralazine: 14.0 ± 1.1 ml/min per g body wt). Although triglyceride levels were not different, UNx mice had significantly higher serum cholesterol when they were killed than sham-operated mice. This elevated cholesterol level found in UNx mice was not affected by losartan treatment. By contrast, hydralazine-treated mice had lower serum cholesterol (Table 1).


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  		Table 1. Systemic parametersa

 
Quantification of Atherosclerotic Lesions
Atherosclerotic lesion size in the proximal aorta was significantly greater in UNx mice versus sham (UNx 251,726 ± 21,881 versus sham 180,299 ± 15,311 µm2; P < 0.05; Figure 1). Losartan treatment significantly lessened UNx-induced acceleration of atherosclerosis (162,865 ± 16,963 µm2 [P < 0.01] versus UNx [NS] versus sham). By contrast, hydralazine treatment provided no such benefit (256,670 ± 10,121 µm2; NS versus UNx, and P < 0.05 versus sham or UNx+losartan).


Figure 1
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  		Figure 1. Cryosections of proximal aortae in sham-operated apolipoprotein E–deficient (apoE–/–) mice (sham), uninephrectomized apoE–/– mice (UNx), UNx mice that were treated with losartan, and UNx mice that were treated with hydralazine. Graph shows the quantitative data of the four groups. *P < 0.05 versus sham; §P < 0.05 versus UNx; {dagger}P < 0.05 versus UNx+losartan. Magnification, x400.

 
Quantitative analysis of en face sections that were stained with Sudan IV revealed that, similar to the proatherogenic effect within the proximal aortic region, UNx amplified atherosclerosis along the entire length of the aorta. Moreover, as in the proximal lesions, the extent of this atherosclerosis was dramatically ameliorated by treatment with losartan but not hydralazine. Thus, en face lesions occupied 1.4 ± 0.5% of the aorta in sham, 4.1 ± 0.4% in UNx (P < 0.05 versus sham), 1.1 ± 0.1% in UNx+losartan (P < 0.05 versus UNx and NS versus sham), and 9.2 ± 1.8% in UNx+hydralazine (P < 0.05 versus sham, UNx, and UNx+losartan; Figure 2). The number of individual plaques was also decreased by losartan but not by hydralazine (sham 23.0 ± 4.3 versus UNx 26.1 ± 1.0 [NS]; UNx+losartan 14.0 ± 1.3 [P < 0.05 versus sham or UNx]; and UNx+hydralazine 21.8 ± 2.3 [NS versus sham UNx and P < 0.05 versus UNx+losartan]; Figure 2).


Figure 2
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  		Figure 2. Representative pictures of en face aortae from sham, UNx, UNx+losartan, and UNx mice that were treated with hydralazine (UNx+hydralazine). Graph shows the quantitative data of the number of individual lesions in en face preparations for the four groups. *P < 0.05 versus sham; §P < 0.05 versus UNx; {dagger}P < 0.05 versus UNx+losartan.

 
Phenotype of Atherosclerotic Lesions
Immunohistochemical assessment for macrophage content that was assessed by MOMA-2 staining revealed that UNx significantly expanded the proportion of the atherosclerotic lesion that is macrophage positive (Figure 3). Thus, whereas MOMA-2 staining occupied 56.3 ± 6.2% of the lesion in sham, this parameter was increased in UNx to 76.3 ± 3.8% (P < 0.05 versus sham). In UNx+losartan, the area occupied by macrophages decreased dramatically to 50.5 ± 5.4% (P < 0.05 versus UNx and NS versus sham). The macrophage area was not decreased by hydralazine (72.5 ± 6.1%; NS versus UNx and P < 0.05 versus UNx+losartan and sham). To test directly the specific effects of losartan and hydralazine on macrophages, we performed in vitro assessments of macrophage migration. In vitro macrophage migration was decreased by losartan but not by hydralazine. Thus, compared with migration of cells that were exposed to Ang II (10–6 M; 26.9 ± 2.2 cells per high-power field), cells that were exposed to Ang II together with losartan decreased macrophage migration by approximately 50% (13.7 ± 1.8 cells per high-power field; P < 0.01 versus Ang II alone). By contrast, there was virtually no decrease in macrophage migration in cells that were exposed to Ang II together with hydralazine (25.4 ± 2.8 cells per high-power field; NS versus Ang II alone; Figure 4). Similarly, a lower dose of Ang II (10–7 M) together with losartan significantly decreased macrophage migration by approximately 25% (P < 0.05), whereas hydralazine was again ineffective in altering migration in Ang II–exposed cells.


Figure 3
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  		Figure 3. Mean area occupied by immunocytochemical staining for macrophages (MOMA-2) in the lesions of sham, UNx, UNx+losartan, and UNx+hydralazine groups. *P < 0.05 versus sham; §P < 0.05 versus UNx; {dagger}P < 0.05 versus UNx+losartan.

 

Figure 4
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  		Figure 4. Migrating activity of peritoneal macrophages that were exposed to angiotensin (AII) together with losartan or hydralazine.

 
Further characterization of the lesions revealed that UNx-associated acceleration in atherosclerosis markedly increased the number of breaks in the elastin of the ECM. Compared with sham, the number of elastin breaks increased from 22.4 ± 2.5 to 39.6 ± 4.1 breaks/mm2 in UNx (P < 0.05). Losartan but not hydralazine treatment dramatically lessened this damage (27.2 ± 2.1 breaks/mm2 in UNx+losartan [P < 0.05 versus UNx] and 37.3 ± 2.8 breaks/mm2 in UNx+hydralazine [NS versus UNx and sham and P < 0.05 versus UNx+losartan]; Figure 5). Notably, immunostaining for the proteolytic enzyme cathepsin S, which was shown recently to have particular relevance to elastin damage and atherosclerosis, clearly was reduced by losartan but not by hydralazine treatment (Figure 6). In contrast, medial thickening was similarly affected by losartan and hydralazine, proportionate to their equivalent effects to decrease BP (Table 1). Thus, medial area was 85,000 ± 6078 mm2 in sham versus 89,317 ± 2723 mm2 in UNx, both significantly higher than those observed in UNx+losartan (71,989 ± 2535 mm2) and in UNx+hydralazine (74,026 ± 1181 mm2).


Figure 5
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  		Figure 5. Verhoeff-van Gieson staining for elastin in sham, UNx, UNx+losartan, and UNx+hydralazine. The graph shows quantitative data of elastin breaks assessed over the entire aorta from the four groups. *P < 0.05 versus sham; §P < 0.05 versus UNx; {dagger}P < 0.05 versus UNx+losartan. Magnification, x400.

 

Figure 6
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  		Figure 6. Immunohistochemical staining for cathepsin S in aortae of UNx (top), UNx+losartan(middle), and UNx+hydralazine (bottom). Magnification, x400.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study confirms that reduction in renal mass, even though not elevating serum creatinine, markedly increases atherosclerotic lesions throughout the aorta. Acceleration in disease was dramatically lessened by Ang II antagonism with losartan but not by a nonspecific antihypertensive, hydralazine. Reduction in the atherosclerotic burden was accompanied by changes in plaque composition toward a lesion that contained less lipid, fewer infiltrating cells, and preservation of the elastin component of ECM together with a decrease in the proteolytic enzyme cathepsin S. These results suggest that even subtle deterioration in renal function amplifies atherosclerotic vasculopathy. These effects of kidney dysfunction can be reduced drastically by Ang II antagonism through mechanisms that include reduced macrophage infiltration and reduced degradation of the ECM component, elastin, which promotes remodeling of the lesion toward a more stable phenotype.

Increased atherosclerosis after UNx was noted along the entire length of the aorta, as assessed by cross-sectional analysis of the proximal aorta and en face preparations of the distal aorta. However, reduction in renal mass did not increase the number of individual plaques, suggesting that, over the time course of observation, UNx had a greater impact on potentiating expansion of existing lesions rather than initiation of new plaques. Increased lesions in proximal cross-sections as well as in distal en face specimens were dramatically reduced by losartan such that at the end of the experiment, the atherosclerotic burden of UNx+losartan at both sites was indistinguishable from the lesions of sham. These results reiterated the beneficial effects of Ang II antagonism on atherosclerosis that was observed in animals with intact kidneys (28,29) and in clinical studies with angiotensin-converting enzyme inhibitors in individuals with intact renal function (13–15). It is of interest that the number of individual lesions in UNx+losartan was not only lower than in UNx but also lower than in sham with intact kidney tissue and suggests the possibility that antagonism of Ang II actions can even reverse the atherosclerotic injury. In stark contrast, none of these benefits was observed in hydralazine-treated mice that had atherosclerosis indistinguishable from untreated UNx mice. Although the data are in good agreement with recent findings that atherosclerotic progression increases as renal mass decreases (16–20), these studies make the novel observation that Ang II is an important mechanism for reduced renal mass acceleration in atherosclerosis. This is of interest because heightened Ang II prevails in CKD, yet there is very little information about therapy for coronary artery disease in CKD, including treatment with Ang II blockers (7). Notably, a post hoc subgroup analysis of the Heart Outcomes Prevention Evaluation (HOPE) study, which described Ang II inhibition in the general population, found that patients with early CKD were especially susceptible to benefits of an angiotensin-converting enzyme inhibitors (30). These findings are especially noteworthy because Ang II antagonism is the premiere medical therapy for progressive renal dysfunction for which heightened Ang II is thought to promote various pathophysiologic mechanisms that are relevant in initiation and progression of atherosclerosis (8–15).

UNx increased BP in our study, which may have contributed to greater atherosclerosis. However, previous studies found similar UNx-induced potentiation in atherosclerosis when pressure was not elevated (17,18). Indeed, our data support that atherogenesis does not depend on systemic hemodynamics in that, although both losartan and hydralazine decreased BP equally, only losartan resulted in decreased atherosclerosis. Nonetheless, it is possible that the intermittent tail-cuff measurements did not capture subtle or diurnal differences in BP between losartan- and hydralazine-treated mice. Similar to recent studies of apoE–/– mice (17,31), UNx did not significantly affect the serum creatinine or creatinine clearance or cause appreciable renal injury within the remaining kidney. However, in view of the potential noncreatinine chromogens confounding mice serum creatinine measurements, it remains possible that serum creatinine and, therefore, the calculated clearance do not accurately reflect the whole-kidney GFR. Thus, although the whole-kidney GFR does not suggest profound decrease in total renal function, it is likely that subtle changes in intrarenal hemodynamic filtration and tubule functions already documented in uninephrectomized animals and humans exist and have an impact on disease process (32,33). Indeed, UNx per se is known to amplify superimposed renal injury (34). Our study confirms that UNx also accelerates injury in other vascular beds.

Serum cholesterol but not triglycerides was increased in UNx mice compared with sham. This hyperlipidemic effect was noted previously without affecting the VLDL, IDL/LDL ratio of HDL levels (17). Although these findings underscore the idea that renal dysfunction–associated dyslipidemia may contribute to CVD, the protective effects of Ang II antagonism occurred in the absence of any reduction in serum hyperlipidemia. Thus, the findings rather underscore the increasingly recognized dissociation between serum lipids and atherosclerotic complications (35). Notably, a recent study of uremic patients found that treatment with cholesterol-lowering statins was only mildly beneficial (36). Instead, the findings emphasize local dynamics within the vascular wall as potentially important modulators of atherosclerosis, including the possibility that benefits of Ang II antagonism in CKD is not dependent on lowering serum lipids.

Examination of the atherosclerotic lesions revealed greater macrophage-positive areas in UNx mice than in sham (Figure 3). Whereas previous studies found macrophages within the lesions of mice with reduced renal mass (17,18), our experiments were specifically designed to provide quantitative data of macrophage content of atherosclerotic lesions. Thus, macrophage content was assessed in the susceptible proximal aorta, at a stage of lesion development characterized by maximal prominence of monocyte-derived cells, by MOMA staining that has been used extensively to quantify the macrophage positivity of the lesion (16,23–25,29,37–40). Our data complement the recognized pivotal role of macrophages in initiation and progression of atherosclerosis in mice with intact kidneys and complements our previous observations regarding the role for Ang II in this process (21). We previously showed that Ang II infusion increased the macrophage content in aortae of mice reconstituted with either proatherogenic apoE–/– or wild-type bone marrow as well as increased migration of macrophages that were exposed to Ang II (21). In another study, we showed that even transient exposure to Ang II increased aortic macrophage content, a change that preceded increased lipid deposition in the vessel wall (22). Our data reveal that Ang II antagonism but not hydralazine treatment dramatically lessened the MOMA-positive area of aortic lesions. Moreover, in vitro macrophage migration was reduced by losartan but not hydralazine. These findings complement previous observations that suggested that reduction in renal mass can affect macrophage behavior. Those studies observed increased endothelial expression of adhesion molecules, including intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (19), and generalized oxidative stress (18), findings that would predict enhanced monocyte adhesion and migration into the vascular intima. Our study shows that this implied consequence actually occurs and suggests that reduction in renal mass stimulates Ang II–dependent mechanisms that promote macrophage infiltration. Such effects on macrophages may contribute to the expanding atherosclerotic lesion while inhibition of Ang II actions may abrogate the proatherogenic effects. It is notable that macrophage movement, specifically emigration, was linked recently to regression of atherosclerosis (39). In addition to modulating the extent of atherosclerosis, macrophage content affects stability of the lesion. Thus, reduction in macrophage content predicts reduction in vulnerability to plaque rupture that may have even greater implications in patients with renal dysfunction.

In addition to amplifying the atherosclerotic lesions within the intima, reduction in renal mass disrupts the vascular morphology within the ECM, including decreased and deranged elastin fibers (41,42). These observations are of interest because of the newly appreciated role of the ECM components in vascular remodeling, including atherosclerosis, where ECM proteolysis is thought to facilitate cellular infiltration and proliferation, angiogenesis, and plaque instability (24,43–46). Notably, whereas the individual components of ECM, including collagen, fibronectin, and laminin, modulate parenchymal response to injury, the previously considered inert elastin seems to be unique in its ability to promote vascular proliferative pathology (43). Our study reveals that UNx per se dramatically increases elastin damage, evidenced by almost doubling in the number of breaks in the elastin lamellae found in UNx mice compared with shams. Losartan but not hydralazine treatment completely abrogated this effect and reiterates previously noted elastolytic properties of Ang II (47). The effects on elastin damage dissociated from UNx effects on the medial area, which was similar in sham and UNx, and decreased similarly with losartan and hydralazine treatment. Although Ang II modulation of matrix metalloproteinase and serine proteases that are capable of degrading ECM components is recognized (48,49), elastin degradation seems especially susceptible to the cathepsin family, particularly cathepsin S (24,50). Recently, hyperlipidemic LDLR–/– mice, which also are deficient in cathepsin S, were found to have significantly less atherosclerosis, together with fewer elastin breaks, compared with LDLR–/– control mice (24). This study finds that along with preserved elastin, cathepsin S in aortae of UNx mice was decreased by Ang II antagonism but not by hydralazine treatment. These findings complement our preliminary studies showing that infusion of Ang II into apoE–/– mice increased elastin breaks and decreased the elastin content of the aortae and that smooth muscle cells that were exposed to Ang II in vitro increased elastolysis (28). In that study, we further showed that cathepsin S mRNA expression was halved in losartan-treated versus untreated mice aortae, correlating with the decreased immunostaining in aortas of mice that were treated with losartan compared with controls. Taken together, these observations suggest that reduction in renal mass stimulates Ang II–responsive mechanisms that promote elastin damage that in turn propagates atherosclerotic lesions, whereas inhibition of Ang II actions abrogates these proatherogenic effects.

In summary, reduction in renal mass that does not induce azotemia potentiates atherosclerosis, which is characterized by greater macrophage-positive area within the atherosclerotic lesions and increased breaks in the elastin component of the ECM. Ang II antagonism decreased atherosclerosis in association with less macrophage content and less elastin damage together with a reduction in the elastolytic cathepsin S. These findings support an important role for endogenous Ang II in accelerated atherosclerosis in renal dysfunction and offer a therapeutic intervention with particular benefit in this setting.


   Acknowledgments
 
This work was supported in part by National Institutes of Health grants DK44757 (V.K.), DK37868 (I.I.), HL53989 (M.F.L), and HL65709 and 57986 (S.F.) and the Lipid, Lipoprotein and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Center (NIH DK59637-01).

We acknowledge the expert technical assistance of Cathy Xu.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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