Immunolocalization of ACE2 and AT 2 Receptors in Rabbit Atherosclerotic Plaques

Materials and Methods

Male New Zealand White rabbits at 3 months of age were randomly allocated into two groups of eight rabbits and fed either a normal rabbit chow diet supplemented with 0.5% cholesterol (Chol) or a normal rabbit chow diet (Con). The animals were housed in individual cages and maintained at a constant temperature of ∼21C. Food and water was supplied ad libitum. The experiments were approved by the Monash University, Department of Anatomy Ethics Committee, and were carried out according to the National Health and Medical Research Council “Australian Code of Practice for the Care and Use of Animals for Scientific Purposes” (6th Edition, 1997).

Immunohistochemistry

Thoracic aortae were processed for paraffin. Paraffin blocks were cut at 5 μm and collected on gelatin-coated slides. Slides were dewaxed, rehydrated, and incubated with blocking serum (1% donkey serum diluted in 10 mM Tris HCl, pH 7.3). Primary antibodies were diluted (AT₂R and ACE2, 1:500 dilution; Santa Cruz Biotechnologies, Santa Cruz, CA) and the slides incubated overnight. Antibody absorbtion was used as a negative control for AT₂R and ACE2. Slides were then rinsed in 10 mM Tris HCl, pH 7.3, and a biotinylated donkey anti-goat polyclonal antibody (Chemicon International; Temecula, CA) was diluted 1:100 in antibody diluent (Dakocytomation, Australia) and the slides incubated for one hour. Slides were then rinsed in 10mM Tris HCl, (pH 7.3) and incubated with an ultrasensitive streptavidin-peroxidase polymer (Sigma-Aldrich, Australia) diluted 1:100 in antibody diluent (Dakocytomation; Carpinteria, CA) for 1 hr. Diaminobenzidine was used as the substrate.

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Figure 1

Photomicrographs of serial adjacent sections of rabbit thoracic aorta demonstrating angiotensin-converting enzyme 2 (ACE2) and angiotensin II type 2 receptor (AT₂R) immunoreactivity. ACE2 immunoreactivity was not clearly evident in normal thoracic aorta endothelia (A), but clearly visible in neointima (C) and plaques (E,G). However, AT₂R immunoreactivity was clearly evident in normal thoracic aorta endothelia (B), as well as in neointima (D) and plaques (F,H). In plaques, both AT₂R and ACE2 immunopositive cells were clearly present and abundant (E-H), and in a magnified area (dashed box) of E and F, smooth muscle cells/myofibroblasts show distinct reactivity for both proteins (dashed circles, G,H). There were also some cells that were positive for AT₂R but not ACE2 (arrows, G,H). (Original magnification: A-D = X300; E,F: X200; G,H: X400).

Monoclonal antibodies to RAM-11 and αSMC actin (Dakocyotmation) were used to identify macrophages and smooth muscle cells/myofibroblasts. In this case, immunohistochemistry was performed using the Envision Kit (Dakocytomation) as previously described (Zulli et al. 2003, 2005).

Results

AT₂R, but not ACE2, immunoreactivity, was clearly visible on the endothelial layer overlying normal vessel wall (Figure 1B and 1A, respectively). However, both proteins were present on the endothelial cell layer overlying neo-intima and atherosclerotic lesions (Figure 1D and 1C, respectively). Both ACE2 and the AT₂R were present in oval and spindle-shaped cells within plaques, as well as in macrophages (RAM-11 positive) both overlying and within atherosclerotic plaques (Figures 11-E1). A magnification of Figures 1E and 1F (dashed box) clearly shows that the majority of cells in plaques were positive for both proteins (dashed circles); however, there were some cells that were only positive for AT₂R, but not ACE2 (Figures 1G and 1H depicted by arrows).

To determine whether macrophages or αSMC actin-positive cells expressed both ACE2 and AT₂R, we used serially sectioned adjacent sections and performed immunohistochemistry for all four antibodies. We observed that a high proportion of macrophages and SMC actin-positive cells within atherosclerotic plaques expressed both ACE2 and AT2R, depicted by circles (Figure 2).

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Figure 2

Photomicrographs of serial adjacent sections of rabbit thoracic aorta demonstrating (A) angiotensin-converting enzyme 2 (ACE2), (B) angiotensin II type 2 receptor (AT₂R), (C) macrophage, and (D) α smooth muscle cell actin positive cells. In this atherosclerotic plaque, ACE2 and AT₂R were clearly visible in α smooth muscle cell actin positive cells, which are either smooth muscle cells or myofibroblasts (circles). As well, macrophages (RAM-11 positive cells) overlying the plaques were also immunopositive for ACE2 and AT₂R proteins. Negative controls show no binding (E: Original magnification X200).

Discussion

We have demonstrated by immunohistochemistry that macrophages and αSMC actin-positive cells within atherosclerotic plaques express high levels of ACE2 and AT₂R proteins. In combination with results published previously by other authors, the results presented in this study add to the growing body of evidence that suggests that all the factors of the renin angiotensin system can be coexpressed in atherogenesis.

It has been previously shown that inhibition of ACE (Schuh et al. 1993) or AT₁R (Candido et al. 2002) with selective pharmacotherapy greatly reduces atherosclerosis. Although the mechanisms of decreased atherogenesis in this model has been suggested to be because of a decrease in AngII formation and AT₁R inhibition, other atheroprotective mechanisms may be stimulated. For example, ACE inhibitors do not inhibit ACE2 activity. Thus it could be suggested that ACE inhibition may not only decrease AngII formation, but also leave angiotensin I to form Ang(1–9) via ACE2, and as we have shown, ACE2 is highly expressed in atherosclerotic plaques. Although there is no direct evidence to suggest that Ang(1–9) is atheroprotective, Ang(1–9) is converted to Ang(1–7), which has antiatherogenic properties (Osei et al. 1993; Ferrario 1998).

Another possible atheroprotective mechanism of AT₁R blockade could be that “free” AngII could bind to the AT₂R. Stimulation of the AT₂R has been shown to inhibit neo-intimal formation (Janiak et al. 1992), decrease smooth muscle cell proliferation (Nakajima et al. 1995) and migration (Chassagne et al. 2002), and inhibit fibroblast proliferation (Tsuzuki et al. 1996). Moreover, AngII can be converted to Ang (1–7) by ACE2 (Boehm and Nabel 2002). Thus we suggest that in the development or regression of atherosclerosis, blockade of the proatherogenic actions of ACE or the AT₁R could unmask the inhibitory actions of ACE2 and the AT₂R.

In conclusion, ACE2 and AT₂R were identified in macrophages and smooth muscle cells/fibroblast within atherosclerotic plaques and the majority of cells expressed both proteins. Although the role of ACE2 and the AT₂R in atherogenesis is suggested to be atheroprotective, the specific role of these factors in atherogenesis warrants further study.