The present study provides evidence that AT2 and AT4 receptors have opposite effects on vascular alteration caused by streptozotocin-induced diabetes in mice, a finding that provides new light on the complex role of the renin-angiotensin- system on the diabetes-induced vascular alteration, and may provide new therapeutic tracks for optimizing vascular prevention in diabetes patients.
Diabetes is associated with both microvascular and macrovascular diseases, manifested as altered vascular morphology and function. Thus, vascular smooth muscle cells (VSMCs) exposed to elevated glucose  and aortic smooth muscle cells from diabetic rabbits or rats exhibit increased in vitro growth rates . Endothelial dysfunction is an early feature of diabetic vascular disease, characterized by a decrease in NO bioavailability and a concomitant increase in vascular O2•- formation . Loss of NO bioavailability precedes the development of overt atherosclerosis and is an independent predictor of adverse cardiovascular events [26, 27].
In our study, induction of diabetes with streptozotocin in young mice induced the well-recognized vascular alterations, with a progressive dysfunction of endothelium-dependent relaxation and hypertrophy of the media in both aortic and mesenteric vascular beds, peaking after 6 weeks of diabetes duration. In this classical experimental model, we demonstrate that treatment with AngIV prevented the functional endothelial alteration and the vascular hypertrophy induced by diabetes. Moreover, when implemented in mice with already established vascular alteration, treatment with AngIV dose-dependently restored functional and morphologic vascular integrity. AngIV did not mediate its favorable actions via effects on metabolic or hemodynamic pathways such as glucose or blood pressure, respectively, two major pathways in the pathogenesis of diabetic vascular disease [28, 29]. Consistent with the observed antitrophic effect, direct measurement by electron paramagnetic resonance showed that AngIV restored a normal balance between the content of NO and superoxide anion in the aortic and mesenteric walls. Taken together, our results fully confirm, and extend to the model of experimental type 1 diabetes the beneficial effect of chronic AngIV treatment first reported by Vinh et al. in Apo-E deficient mice , and further establish that AngIV acts by increasing NO bioavailability and decreasing oxidative stress.
However, our findings yield opposite conclusions as regards the respective contribution of AT2 and AT4 receptors in mediating the actions of AngIV. In the Apo-E deficient mice model, the protective effect of AngIV on endothelial dysfunction was attenuated by both the specific AT4 antagonist Divalinal and the AT2 antagonist PD123319, suggesting involvement of both receptors . In a previous work, our group has reported that AngIV was protective in a model of acute ischemic stroke, and that both AT2 and AT4 antagonists likewise inhibited this protective effect . In contrast in our diabetic mice, Divalinal completely blunted the protective effect of AngIV on EDR, whereas PD123319 had no effect. To further examine the role of the AT2 receptor, we thus studied the effect of AT2 pharmacological blockade in the absence of AngIV, and found that PD123319 had no effect on EDR in control mice, but completely blunted the diabetes-induced alteration of EDR. In the ApoE-deficient mice, AngIV blunted the increase of dihydroethidium staining for superoxide and PD123319 significantly inhibited this effect of AngIV. In sharp contrast we found that both AngIV and PD123319 equally inhibited diabetes-induced increase of superoxide production. Thus, the protective effect of AngIV in the atherogenic model of ApoE-deficient mice appears to be mediated by both AT2 and AT4 receptors, whereas in streptozotocin-induced diabetes mice, the protective effect of AngIV was solely mediated by AT4, while AT2 receptor stimulation seemed to play a detrimental role. This puzzling observation led us to extend our study and to further examine the role of AT2 by studying the effect of diabetes on vascular alterations in genetically modified mice lacking the AT2 receptor. In full consistence with the results gained with the pharmacological blockade of the AT2 receptor, we found that AT2 null mice were fully protected against the endothelial dysfunction and the vascular hypertrophy of the mesenteric arteries induced by six weeks of diabetes. In a study performed in ApoE/ AT1A receptor double knockout mice , chronic AT2 receptor inhibition with PD123319 increased plaque development, whereas direct AT2 receptor stimulation reduced atherogenesis, demonstrating, in accordance with Vinh et al., an antiatherosclerotic role of the AT2 receptor. However, Koitka et al. reported that in ApoE-deficient mice, induction of diabetes with streptozotocin increased the aortic expression of the gene Agt2r, and was associated with a six-fold increase in plaque area that was significantly attenuated by both AT2R pharmacological blockade and AT2R deletion.
These patent discrepancies regarding the role of the AT2 receptor, that appears to be protective in the ApoE-deficient model of accelerated atherosclerosis, but detrimental in the setting of diabetes in fact no really surprising, and adds to a long list of controversial data. Left ventricular hypertrophy (LVH) is a major predictor of cardiovascular morbidity and mortality, and it is unanimously accepted that the angiotensin AT1 receptor is involved in the pathogenesis of hypertension and LVH, but the role of the AT2 receptor in LVH is still controversial. Studies addressing the involvement of the AT2 receptor in LVH performed in genetically altered, either AT2 receptor-deficient or AT2 receptor-overexpressing yielded highly controversial results with an equal number of studies supporting prohypertrophic, antihypertrophic, or neutral effects of the AT2 receptor in LVH .
These discrepancies have been discussed in deep elsewhere , and support the view that the mysterious versatility of the AT2 receptor phenotype appears to be highly cell-type and tissue specific, but also to depend on the autocrine/paracrine regulation of the cellular milieu of the target organs, and the specific experimental or pathophysiological conditions that determine the complex cross-talk between AT1 and AT2 receptors. For instance, You et al.  have shown that high blood pressure reversed the classical AT2 receptor-mediated vasodilation into vasoconstriction in spontaneously hypertensive rats, in agreement with a previous study showing that in young hypertensive rats AngII-induced contraction was decreased by AT2R blockade . The mechanism of this reversal remains to be discovered, but may involve a switch in signaling from constrictor to dilator mechanisms due to increased endothelial AT2R expression. AT2R-dependent contraction in SHR is not affected by endothelium removal whereas AT2R-dependent dilation is abolished in the absence of endothelium. Thus the difference in the type of response might reflect a change in AT2R expression between the endothelium and the smooth muscle.
A recent study tested the effects of a 2 weeks treatment with the AT2 receptor agonist CGP-42112A on inflammation and oxidative stress in obese Zucker rats and compared them to their lean counterparts: the results suggested anti-inflammatory and antioxidative functions of AT2 receptor in obese Zucker rats, but proinflammatory and prooxidative functions in lean Zucker rats  further suggesting that AT2R function depends on the pathophysiological context.
In our study, diabetes was induced by streptozotocin, a convenient and widely used experimental model that is not devoid of criticisms and limitations. Noticeably, streptozotocin induces diabetes, but also elicits a strong and sustained inflammatory state. It is thus clearly possible that the deleterious effect of AT2R herein reported is the consequence of streptozotocin-induced side effects rather than that of diabetes per se, and the potential role of AT2R needs to be further studied in other experimental models of diabetes. Nonetheless, the observation that, in given experimental conditions, the AT2 receptor stimulation has a deleterious effect on vascular functional and morphological integrity challenge the concept that AT2 receptor stimulation represents the counter-regulatory arm of the RAS that unequivocally balances and opposes the effects of AT1 stimulation. Indeed, in spite of the uncertainties and discrepancies regarding AT2 functions, the view has emerged that the physiological functions of AT2 receptor is to antagonize the effects of the AT1 receptor , and that novel strategies to improve cardiovascular diseases may rely on drugs activating the tissue-protective arms of RAAS [39, 40] The discovery of a first non-peptide, orally active AT2-receptor agonist compound 21 (C21), that should enter soon the first steps of clinical studies will help to clarify the therapeutic potential of AT2 stimulation , but our findings stress the need to cautiously delineate the specific pathological conditions in which it may prove beneficial, or in contrary may reveal potentially harmful.
During the last decade, AT4 has been identified as insulin-regulated aminopeptidase (IRAP) . The nature of the molecular mechanism by which this membrane-bound enzyme mediates the variety of intracellular signaling and the biological effects triggered by AngIV remains so far obscure, and several hypotheses are still debated (see  for review). Interest on AngIV/AT4 (IRAP) pathway has nonetheless been boosted by its potential ability to enhance cognitive function and memory . This potential interest as prompted active ongoing research to identify small biologically active non-peptides molecules that, like AngIV, inhibit the catalytic domain of IRAP . Such new AT4 agonists will pave the way for innovative research for the prevention of cognitive decline, but also for stroke therapy [30, 46, 47]. Clearly our present findings, confirming the vascular protective effect of AngIV in type 1 diabetes mice also invite to consider AT4/IRAP as another potential target for revisited therapeutic strategies of RAAS modulation for cardiovascular disease prevention.