Overexpression of ACE2 Enhances Plaque Stability in a Rabbit Model of Atherosclerosis
Objective— The purpose of this study was to test the hypothesis that ACE2 overexpression may enhance atherosclerotic plaque stability by antagonizing ACE activity and converting angiotensin II to angiotensin 1–7.
Methods and Results— Atherosclerotic plaques were induced in the abdominal aorta of 114 rabbits by endothelial injury and atherogenic diet. Gene therapy was performed in group A at week 4 and in group B at week 12, respectively. Each group of rabbits were randomly divided into 3 subgroups which received, respectively, a recombinant ACE2 expressing vector (AdACE2), a control vector AdEGFP and AdACE2+A779, an antagonist of angiotensin 1–7 receptor. Local ACE2 overexpression attenuated the progression of lesions from week 4 to week 8, but not progression of plaque size from week 12 to week 16. In group B rabbits, local ACE2 overexpression resulted in stable plaque compositions, ie, fewer macrophages, less lipid deposition and more collagen contents, higher plaque stability scores, decreased angiotensin II levels, and increased angiotensin 1–7 levels in plaque tissues in the AdACE2 subgroup compared with those in the AdEGFP subgroup.
Conclusions— Overexpression of ACE2 results in stabilized atherosclerotic plaques and the mechanism is probably the conversion of vasoconstrictive angiotensin II to vessel protective angiotensin 1–7.
Recent studies have shown that the endogenous levels of angiotensin II (Ang II) are regulated by the opposing action of 2 carboxypeptidases, angiotensin-converting enzyme (ACE) and ACE2. The latter is a more recently discovered homologue of ACE and is thought to counterbalance ACE by cleaving Ang I and Ang II into inactive Ang 1–9 and vasodilating and antiproliferative Ang-(1–7), respectively. ACE2 is thus considered a potential therapeutic target of the rennin-angiotensin system (RAS) for treatment of cardiovascular diseases owing to its key role in the formation of vessel protective peptides from Ang II.1,2
Both ACE and ACE2 are considered key regulators of many cardiovascular pathological processes. Although Ang II and its receptor angiotensin subtype 1 receptor (AT1R) have been reported by many studies to be expressed in atherosclerotic lesions, ACE2 was reported only recently to be expressed in vascular endothelial cells, macrophages, and smooth muscle cells (SMCs).3 More recently, ACE2 gene transfer was reported to result in a significant regression of left ventricular hypertrophy in spontaneously hypertensive rats.4 However, little is known about the exact role of ACE2 in the formation and stabilization of atherosclerotic plaques. Because local RAS plays an important role in the pathogenesis of atherosclerosis,5 it is reasonable to assume that imbalance of the activities of these 2 enzymes, ACE and ACE2, may have paramount importance in the pathogenesis of atherosclerosis. Therefore, we hypothesize that overexpression of ACE2, through counteracting with ACE and degrading Ang II into Ang-(1–7), may tip the balance of RAS expressions and stabilize atherosclerotic plaques. Based on this hypothesis, we determined the effects of ACE2 overexpression via an adenovirus vector on aortic plaques and a number of parameters in relation to plaque stability in a rabbit model of atherosclerosis.
Materials and Methods
Detailed material and methods are described in the supplemental data (available online at http: //atvb.ahajournals.org).
Preparation of ACE2 Adenovirus Vectors
The murine ACE2 cDNA was amplified by RT-PCR from RNA of mouse kidney. Recombinant adenoviruses (Ad) carrying the murine ACE2 (AdACE2) or a control transgene EGFP (AdEGFP) were prepared as previously described with the AdMax system (Microbix Biosystems).6,7
Animal Model and Gene Transfer
One hundred fourteen New Zealand White rabbits underwent high-fat feeding and balloon-induced arterial wall injury at the beginning of the experiment after anesthesia using a previously described method.8 The rabbits were then divided into group A (n=57) and group B (n=57), which were fed atherogenic diet for 8 and 16 weeks, respectively. At the end of week 4 and week 12, laparotomy was conducted in group A and group B rabbits, respectively, and each group was then divided into 3 subgroups randomly. The first subgroup of rabbits (AdACE2 subgroup, n=19) received infusion of AdACE2 suspension (2.5×109 pfu/mL), the second subgroup (AdEGFP subgroup, n=19) received the control vector AdEGFP, and the third subgroup (AdACE2+A779 subgroup, n=19) received infusion of AdACE2 suspension (2.5×109 pfu/mL) and A779 (Bachem), an Ang-(1–7) receptor antagonist at a dose of 200 ng kg−1 min−1 for 28 days. Four weeks after gene therapy, rabbits in group A and group B were euthanized at the end of week 8 and week 16, respectively, and their aortas were collected for pathological and biochemical analysis.
SMC Cell Culture and Transfection With AdACE2
SMCs from the abdominal aorta in rabbits fed with a normal diet were cultured and were used to determine the infection and expression efficiency of AdACE2.
Measurement of ACE2 and ACE Protein Expression and Activities
ACE2 and ACE protein expression from membrane and total proteins of SMCs and plaque tissue were detected by Western blot,9 and enzymatic activity of ACE2 and ACE in aortic plaques was evaluated by surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS).10
Quantification of Ang II Concentration in Atherosclerotic Plaques by ELISA
The concentration of Ang II in atherosclerotic plaques in group B was measured by a commercial ELISA kit (SPI-BIO).11 The tissue concentration of Ang II is expressed as the Ang II content per 1 mg of protein.
THP-1 Cell Line Culture, Transfection, and ELISA
To examine whether ACE2 overexpression will result in a different response to Ang II in terms of MCP-1, matrix metalloproteinase (MMP)-3, MMP-9, and TIMP-1 release, the THP-1 cells were cultured and transfected by AdACE2 and then ELISA was performed.
Biochemical Studies and Blood Pressure Measurement
The serum levels of total cholesterol (TC), triglyceride (TG), soluble vascular cell adhesion molecule-1 (sVCAM-1), high sensitivity C-reactive protein (hsCRP), and monocyte chemoattractant protein 1(MCP-1), and both systolic (SBP) and diastolic blood pressures were measured.
Histology and Immunohistochemistry
Serial sections were stained with hematoxylin and eosin (H&E), oil red O, and Masson trichrome. Macrophages, SMCs, MCP-1, and ACE2 were identified using appropriate primary antibodies. The plaque stability score was calculated as previously described.12,13
Zymography and Western Blot
The activity of MMP-3 and MMP-9 in the aortic plaques was evaluated by zymography,14 and the protein expression of MCP-1, TIMP-1, and Ang-(1–7) in plaque tissue was assayed by Western blot, respectively.
Gene Expression Analysis
The gene expression levels of matrix Gla protein (MGP), heat shock protein 47 (hsp47), collagen type I, collagen type III, MCP-1, ACE2, and TIMP-1 were quantitatively analyzed using RT-PCR.
Data analysis involved use of SPSS 11.5. All data were expressed as means±SD. Independent sample t test was used to compare continuous data for between group differences, and paired t test was applied to within animal comparisons at different time points. A probability value <0.05 was considered statistically significant.
Plaque Morphology After ACE2 Gene Transfer
In the abdominal aortic segments of group A animals (Figure 1A, upper panel), marked intimal thickening was present in the AdEGFP subgroup. However, intimal thickness, intimal/media thickness ratio (I/M thickness), intimal area, and intimal/media area ratio (I/M area) were markedly lower in the AdACE2 subgroup than in the AdEGFP subgroup (P<0.05). Conversely, with the administration of A779 in Ad.ACE2+A779 subgroup, the intimal thickness, I/M thickness ratio, intimal area, and I/M area ratio increased respectively, compared with those in AdACE2 (P<0.05) subgroup (supplemental Table I). This result indicates that ACE2 overexpression attenuates the progression of early atherosclerotic lesions from week 4 to week 8 in a rabbit model of atherosclerosis. In contrast, in the abdominal aortic segments of group B (Figure 1A, lower panel), the intima thickness, intimal/medial thickness ratio (I/M), the intimal area, and the intima to media area ratio (I/M area) were not statistically different among the 3 subgroups of rabbits (supplemental Table II), suggesting that ACE2 overexpression has no effects on the progression of plaque size from week 12 to week 16.
ACE2 Expression and Activity After Gene Transfer
To determine the efficiency of AdACE2 gene transfer, the expression of ACE2 mRNA and protein were first evaluated in AdACE2 transfected cultured SMCs obtained from the abdominal aorta in rabbits fed with a normal diet. At 3 or 7 days after transfection with 100 MOI of AdACE2 or AdEGFP, ACE2 mRNA was detected by RT-PCR in AdACE2 or AdEGFP transfected or PBS treated SMCs (supplemental Figure 1A). ACE2 protein was identified at ≈80 kDa, the anticipated size of murine ACE2, by Western blot analysis in the AdACE2 or AdEGFP transfected or PBS treated SMCs extracts (supplemental Figure IB).
The expression of ACE2 mRNA and protein in plaques were determined in group B at week 16. ACE2 mRNA level was significantly higher in AdACE2 or AdACE2+A779 subgroups than in AdEGFP subgroup (supplemental Figure IV). Immunohistochemical staining displayed that ACE2 protein was highly expressed in plaques of the AdACE2 or AdACE2+A779 subgroup but only weakly expressed in plaques of AdEGFP subgroup (Figure 2A). Western blot analysis demonstrated that ACE2 proteins extracted from membrane or total proteins both exhibited significantly higher expression levels in AdACE2 or AdACE2+A779 subgroup than in the AdEGFP subgroup (supplemental Figure II).
To determine whether macrophages and αSM actin–positive cells express ACE2 protein, adjacent sections were used for immunohistochemical staining of antibodies against RAM-11, a-SM actin, and ACE2. The results showed that a large proportion of macrophage-positive cells within atherosclerotic plaques expressed ACE2 protein (Figure 2B). In contrast, only a small percentage of αSM actin–positive cells expressed ACE2 protein (Figure 2B).
For ACE2 activity determination, plaque extracts from 3 subgroups in group B rabbits (Figure 3A) or transfected SMCs (supplemental Figure IC) were respectively incubated with Ang II (10 μmol/L) and then detected by SELDI-TOF-MS. In AdACE2 subgroup, marked conversion of Ang II (1046 m/z) to Ang-(1–7) (899 m/z) was evident, providing direct evidence that ACE2 activity in atherosclerotic plaque or SMCs were increased by AdACE2 transfection as expected. In contrast, little Ang-(1–7) (899 m/z) was formed in plaque extracts in the AdEGFP subgroup, indicating that the endogenous ACE2 activity was at a low level. In addition, little ACE2 activity was observed in AdEGFP transfected SMCs. These data suggest that transfection with AdACE2 may enhance ACE2 enzyme activity both in vitro and in vivo.
The level of peptide peaks of Ang-(1–7) and Ang II in the total protein of the aortic plaques in group B was measured by SELDI-TOF-MS. The level of Ang-(1–7) was higher in AdACE2 (55.85±7.97m/z) and AdACE2+A779 (56.00±5.44 m/z) subgroups than that in AdEGFP (17.06±4.89 m/z) subgroup (P<0.01). In contrast, the level of Ang II was lower in AdACE2 (47.08±1.74 m/z) and AdACE2+A779 (47.17±3.97 m/z) subgroups than that in AdEGFP subgroup (51.83±3.76 m/z) (P<0.05). As expected, there was a marked increase in the ratio of Ang-(1–7)/Ang II in AdACE2 (1.19±0.14) or AdACE2+A779 (1.15±0.11) subgroup compared with that in AdEGFP subgroup (0.21±0.14, P<0.01). Nonetheless, the ratio of Ang-(1–7)/Ang II in the AdACE2 subgroup was lower than that in the recombinant ACE2 protein control group (2.28±0.12, P<0.05). Similar measurements of Ang-(1–7) and Ang II were obtained from the membrane protein of the aortic plaques in group B (supplemental Figure ID).
Furthermore, the level of Ang II and Ang-(1–7) in the abdominal aortic plaques in group B was also measured by ELISA and Western blot, respectively. The levels of Ang II in AdACE2 subgroup (2.76±0.20) and AdACE2+A779 subgroup (2.83±0.20) were lower than that in AdEGFP subgroup (4.26±0.22, both P<0.01) but did not differ between AdACE2 and AdACE2+A779 subgroups. On the other hand, the levels of Ang-(1–7) in AdACE2 subgroup (1.85±0.06) and AdACE2+A779 subgroup (1.81±0.07) were higher than that in AdEGFP subgroup (0.64±0.05, both P<0.01) but did not differ between AdACE2 and AdACE2+A779 subgroups (Figure 3C through 3E). In contrast, the levels of Ang II and Ang-(1–7) in the thoracic aortic plaques in group B were not significantly different among the 3 subgroups of rabbits (supplemental Figure IIIA through IIIC). These results provided evidence that AdACE2 effectively infected the abdominal atherosclerotic plaques and rendered the local tissue to overexpress enzymatically active ACE2 protein that lowered the local Ang II concentration and increased Ang-(1–7) level in vivo.
Effect of ACE2 Gene Transfer on ACE Protein Expression and Activity
As described above, Ang I appeared to be effectively converted to Ang II in plaque extracts from all 3 subgroups in group B rabbits (Figure 3B). However, the ratio of Ang II/Ang I peak was lower in AdACE2 (0.62±0.12) and AdACE2+A779 (0.64±0.10) subgroups than in AdEGFP subgroup (1.12±0.04, P<0.01). The ACE protein expression was higher in the AdEGFP subgroup than in the 2 AdACE2 subgroups, but the levels did not differ between AdACE2 and AdACE2+A779 subgroups (supplemental Figure IV), suggesting that ACE2 overexpression may counteract the action of ACE.
Plaque Inflammation After ACE2 Gene Transfer
As shown in Figure 4A, macrophage infiltration, as indicated by RAM-11 staining in the intima regions of the abdominal aorta, was intense in AdEGFP subgroup but was sparse in the 2 AdACE2 subgroups of group B rabbits. MCP-1 staining was remarkable in the neointima of the arteries in AdEGFP subgroup but was less in the AdACE2+A779 subgroup and more sparse in the AdACE2 subgroup (Figure 4A). The difference between AdACE2 and AdEGFP subgroups in abdominal plaques was significant for both macrophage infiltration (13.57±4. 18% versus 23.61±6. 94%, P<0.01) and MCP-1 staining (13.17±6. 44% versus 25.01±7. 43%, P<0.01; Figure 4B). The lower level of MCP-1 protein in arteries of AdACE2 subgroup was also demonstrated by Western blot (Figure 5A). Administration of A779 resulted in 35% and 31% higher macrophage infiltration (18.32±2.41%) and MCP-1 protein expression (17.32±4.09%), respectively, than those in AdACE2 subgroup (all P<0.05), but the levels were still significantly lower than those in the AdEGFP subgroup.
This reduced inflammation in group B was found to be a local effect because the extent of macrophage infiltration, as well as expression levels of MCP-1 protein, in plaques of the thoracic aorta were not statistically different among the 3 subgroups of rabbits (supplemental Figure IIID and IIIE).
Plaque Collagen Deposition After ACE2 Gene Transfer
In group B, the total number of SMCs in plaques was found to be similar among the 3 subgroups of rabbits (Figure 4A, the third row). Similarly, key parameters of collagen synthesis were not different among the 3 subgroups, including expression of MGP, a surface marker of vascular SMCs synthesizing collagen, and expressions of genes involved in collagen synthesis such as heat shock protein 47 (hsp47), chain 1 of collagen type I, and chain 1 of collagen type III (supplemental Figure IV). The gene and protein expression of TIMP-1 was not statistically different among the 3 subgroups of rabbits (Figure 5B and supplemental Figure IVA). On the other hand, the enzymatic activities of MMP-3 and MMP-9 in the AdACE2 subgroup, as revealed by zymography, were comparable with those in the AdACE2+A779 subgroup but were both significantly lower than those in the AdEGFP subgroup (Figure 5C and 5D).
However, with similar number of SMCs, more collagen was found in the abdominal aorta plaques of the AdACE2 subgroup and the AdACE2+A779 subgroup than in the AdEGFP subgroup. Comparing the 2 AdACE2 subgroups, administration of A779 resulted in approximate 8% decrease in collagen content. In addition, the lipid-positive staining in plaques was more evident in AdEGFP subgroup than in AdACE2 and AdACE2+A779 subgroups (P<0.05, Figure 4A, bottom panel). The plaque stability score calculated was greatest in AdACE2 subgroup, which is significantly higher than that in AdEGFP subgroup (P<0.01, Figure 4C) or AdACE2+A779 subgroup (P<0.05). This score in AdACE2+A779 subgroup was also significantly higher than that in the AdEGFP subgroup (P<0.05).
Systematic Influences as Indicated by Biological Measurements and Blood Pressure
The serum levels of total cholesterol (TC), triglyceride (TG), hs-CRP, sVCAM-1. and MCP-1, and blood pressure at the end of week 16 were not significantly different among the 3 subgroups of group B rabbits (supplemental Tables III and IV).
There were 2 major findings in the present study: First, overexpression of ACE2 in group A rabbits attenuated the progression of early atherosclerotic lesions from week 4 to week 8, but had no effects on the progression of plaque size from week 12 to week 16 in the gene transfected site in group B. Second, ACE2 overexpression in group B reduced local inflammation and lipid deposition and increased collagen contents, resulting in stabilized abdominal aortic plaques. The major mechanism of the observed benefits is probably attributable to reduction of endogenous Ang II and production from Ang II to vessel protective Ang-(1–7). To the best of our knowledge, this is the first study to report that local ACE2 overexpression may enhance the stability of atherosclerotic plaques and local ACE2 overexpression at different stages of plaque formation have differential effects.
The differential effects of ACE2 overexpression between early and late stage plaques might be explained by the changes of ACE expression pattern and local Ang II level in plaque formation and progression. Expression of ACE protein was found to be modest in less complicated lesions and it increases as lesions became more complex and is most prominent in macrophage-rich regions.15 The level of Ang II was found in parallel with ACE expression and positively correlated with the size of plaques, quantity of inflammatory cells (in particular, macrophages), the degree of hyperlipidemia, and monocyte/macrophage differentiation.16–17 Therefore, the same AdACE2 gene therapy could inhibit the process of plaque progression at a early stage but could only stabilize the plaques when given at a later stage. Although the mechanism awaits further validation, our observation on the effects of ACE2 gene therapy at different stages of atherosclerosis progression may have important implications in its further applications.
In the present study, overexpression of ACE2 was accompanied by a marked decrease in MCP-1 expression and macrophage accumulation, and a significant increase in aortic intimal collagen content. Many in vitro studies have demonstrated that Ang II promotes the production of MCP-1 by endothelial cells, smooth muscle cells, and human macrophage cell line THP-1, which were confirmed in the present study (supplemental Table V). Furthermore, Ang II induces the adherence of monocytes to endothelial cells, increases macrophage infiltration, and accelerates the atherogenic process.18–19 Our study showed that overexpression of ACE2 inhibited MCP-1 expression and macrophage accumulation, and these beneficial effects were remarkably blunted by administration of A779. These results demonstrated that overexpression of ACE2 in atherosclerotic plaques exerted a strong antiinflammatory effects locally.
An increased collagen level was observed in plaques of AdACE2 infected rabbits, indicating enhanced plaque stability. To further elucidate the possible mechanisms of this finding, factors related to collagen synthesis and degradation were examined but no significant differences were found among the 3 subgroups of rabbits in all the factors examined in relation to collagen synthesis, including the number of VSMCs, gene expression of MGP, chain 1 of collagen type I, chain 1 of collagen type III, and hsp47. However, for the factors related to collagen degradation, a major difference was observed for MMP-3 and MMP-9 activities, which were markedly lower in the AdACE2 infected than the AdEGFP infected plaques. MMP-3 and MMP-9 have been shown to be upregulated in human atherosclerotic plaques and play an important role in extracellular matrix degradation and rupture of vulnerable plaque.20 A high level of Ang II has been reported to be associated with aortic MMP activation and arterial remodeling, whereas a subpressor level of Ang II reported to enhance arterial collagen deposition.21 Our results demonstrated that overexpression of ACE2 inhibited MCP-1, MMP-3, and MMP-9 activity and expression. In addition, our data lends support to the finding of Wang et al that subpressor level of Ang II may in fact promote collagen deposition.21,22
ACE and ACE2 are a pair of enzymes with sequence homology and close functional connections. They could use each other’s product as its own substrate and produce a product with opposing functions, thus forming a forward negative feedback loop in RAS. Apparently, the balance of the 2 enzymes is critical for the functional state of RAS. In our study, by overexpressing ACE2 with an Ad vector in a tissue where the endogenous ACE2 activity was low, this balance has been shifted toward a lower Ang II concentration with less vasoconstrictive effects. Moreover, our results showed that overexpression of ACE2 resulted in not only decreased Ang II level but also increased Ang-(1–7) production, as well as a lower ACE expression level in plaque tissues of the AdACE2 subgroup compared with the AdEGFP subgroup. The inverse relation between ACE2 and ACE expression has recently been reported in cardiac myofibroblasts.23 Although the exact mechanism underlying this observation is unclear, decrease of ACE expression contributes to further lowering of the Ang II level. These results suggest that plaque stabilizing effects of ACE2 overexpression are most likely attributable to the net effect of decreased Ang II level and increased Ang-(1–7) level.
In summary, our studies showed that ACE2 overexpression in atherosclerotic plaques effectively reduced MCP-1 expression, macrophage accumulation, and MMP-3 and MMP-9 activities, and increased collagen content, leading to greater plaque stability in a rabbit model of atherosclerosis. Administration of A779 to antagonize the Ang-(1–7) receptor significantly attenuated the beneficial effects achieved by ACE2 overexpression. The plaque stabilizing effects of ACE2 overexpression are likely to result from the net effect of shifting RAS balance, which is the result of interactions of a variety of system components, toward decreased Ang II level and increased Ang-(1–7) level.
We thank Hong Jiang, Yue Hui Zhang, Rong Wang, and Xu Ping Wang for their technical assistance.
Sources of Funding
This study was supported by the National 973 Basic Research Program of China (No. 2006CB503803), the National High-tech Research and Development Program of China (No. 2006AA02A406), the Program of Introducing Talents of Discipline to Universities (No. B07035), and grants from the National Natural Science Foundation of China (No. 30470701, 30470702, 30570747, and 30670873).
B.D., C.Z., and J.B.F. contributed equally to this study.
Original received May 16, 2007; final version accepted April 1, 2008.
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