Immunolocalization of ACE2 and AT 2 Receptors in Rabbit Atherosclerotic Plaques

Abstract

Evidence suggests that angiotensin type 2 receptor (AT₂R) and angiotensin-converting enzyme 2 (ACE2) play a protective role in atherogenesis. These factors have not been identified in rabbit atherosclerotic plaques. Our goal was to localize ACE2 and AT₂R in rabbit atherosclerotic tissues, and determine which cell types express these factors. New Zealand White rabbits were fed either a control diet or a 0.5% cholesterol diet (n=8/group) for 12 weeks. Paraffin-fixed thoracic aorta were serially sectioned and processed for immunohistochemistry using commercially available antibodies to ACE2, AT₂R, RAM 11 (to identify macrophages), and α smooth muscle cell actin (αSMC) to identify smooth muscle cells and myofibroblasts. AT₂R immunoreactivity, but not ACE2 immunoreactivity, was clearly present in endothelia overlying normal wall. However, both AT₂R and ACE2 immunoreactivity were clearly present in endothelia overlying neo-intima formation and atherosclerotic plaques. Within plaques, both AT₂R and ACE2 immunoreactivity were observed in macrophages and αSMC actin-positive cells. Examination of serial sections showed that the majority of cells were both ACE2- and AT₂R-positive. Macrophages and αSMC actin-positive cells produce ACE2 and the AT₂R in atherosclerotic plaques. Determining a role for these factors in the control of atherosclerosis will require additional studies.

The renin angiotensin system plays a major role in atherogenesis (Lee et al. 1993). Angiotensin II (AngII), the main effector peptide of this system, can bind to both the AngII type 1 receptor (AT₁R) and the AngII type 2 receptor (AT₂R) (de Gasparo et al. 2000). Accumulating evidence suggests that the AT₂R has an antiatherogenic role, which contrasts the function of AT₁R. For example, in vascular injury, AT₂R is reexpressed in interstitial and neointimal tissues and it is believed to regulate tissue remodeling through an inhibitory effect on Ang II-induced mitogen signaling and enhancement of programmed cell death, countering effects of AT₁R stimulation (Matsubara 1998).

Angiotensin-converting enzyme 2 (ACE2) is a newly described enzyme that can break down angiotensin I into Ang (1–9) and Ang II into Ang (1–7) (Boehm and Nabel, 2002). Although Ang (1–7) may in itself be atheroprotective (Osei et al. 1993; Ferrario 1998), the conversion of AngII to Ang (1–7) via ACE2 can be atheroprotective simply because it removes AngII from the atherogenic milieu.

ACE and AT₁R expression have been previously reported in rabbit atherosclerotic tissues (Schuh et al. 1993). Furthermore, the inhibition of either ACE activity (Candido et al. 2002) or inhibition of AT₁R activation (Li et al. 1999) by selective pharmacotherapy greatly reduces atherosclerosis formation in the rabbit model of atherosclerosis, indicating a detrimental role for ACE and the AT₁R in atherogenesis.

Thus it is hypothesized that in atherogenesis, ACE, ACE2, AT₁R, and the AT₂R are all expressed, and that the inhibition of ACE or AT₁R would leave ACE2 and AT₂R unopposed to impair atherogenesis. However, if credence is to be given to this theory, high levels of ACE2 and AT₂R should be expressed in atherosclerotic tissues, because ACE and AT₁R levels have already been shown. To this end, immunohistochemistry for ACE2 and AT₂R were performed on rabbit atherosclerotic tissues, and specific cell markers were used to identify macrophages and α smooth muscle cell actin (αSMC)-positive cells (smooth muscle cells and myofibroblasts).

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.

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).

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.