Should we be sympathetic to angiotensin II infusion?

Hypertension, or abnormally elevated blood pressure, remains a disorder affecting almost a billion people worldwide. It is found across Western society and is increasing in developing communities. A significant factor in the development of hypertension is ageing, with some two-thirds of people exhibiting elevated blood pressure by the age of 60 years, increasing to ∼80% at 80 years. Furthermore, an association with obesity and type 2 diabetes is likely, and there is an increase in its incidence at younger ages as well as in its overall prevalence. Importantly, the long-term consequences of hypertension lie in it being a significant risk factor in a number of common cardiovascular diseases, including heart attack, stroke, atrial fibrillation and heart failure, as well as renal failure. As such, hypertension is a significant cause of morbidity and mortality, as well as being a considerable economic burden to health systems. While some 5–10% of cases of hypertension can be explained as occurring secondarily to another disorder (for example, pheochromocytoma, hyperaldosteronism, renovascular disturbances) the exact cause for the vast majority of cases remains unknown, probably owing to its multifactorial nature. This is despite a large number of investigations over many years and hypotheses relating to the underlying mechanisms being vigorously debated (see recently published Point: Counterpoint discussion; Esler et al. 2010;Navar, 2010). Amongst numerous mechanisms that have been proposed, two of the most vigorously contested relate to the roles of the renal renin–angiotensin system and activation of the sympathetic nervous system (SNS). However, rather than there being a single aetiology, it is likely that multiple mechanisms may be involved, within an individual or between individuals. For example, links are being shown between angiotensin II (AII), mineralocorticoid receptor activation, fibrosis/remodelling and increases in vascular stiffness (McCurley et al. 2012). These appear to involve both renal and non-renal inputs. Furthermore, the involvement of genetic and epigenetic factors should not be underestimated. Interestingly, the multifactorial basis of hypertension is not a new concept and was stressed in the earlier work of Page and colleagues, as reviewed by Harrison (2013). Other factors contributing to the divergence of opinions in the literature perhaps relate to methodological differences and experimental design. Many studies are necessarily conducted at a single time point, making it difficult to establish cause-and-effect relationships. Many investigations consider only a single mechanism, ignoring interactions that may be occurring between vascular beds and regulatory mechanisms. Furthermore, many studies conducted in vitro can be criticized for studying vessels and organs in the absence of circulating, neural and haemodynamic factors, a point that is stressed by Wang and co-workers in this issue of The Journal of Physiology (Wang et al. 2013). To circumvent some of these limitations and to address more directly whether increased vascular resistance in AII-induced hypertension is dependent on the SNS, Wang et al. (2013) used a sophisticated in vivo imaging approach to determine whether this slowly developing model of hypertension is associated with changes in arterial intracellular Ca2+ and vessel diameter and, if so, what the underlying mechanisms may be. The animal model used in these studies was the mouse expressing an exogenous myosin light chain kinase (exMLCK) biosensor in vascular smooth muscle, originally developed by James Stull's laboratory (Isotani et al. 2004) and later adapted to the in vivo studies of blood vessels by Zhang et al. (2010). This approach enables Förster resonance energy transfer (FRET) to be studied in vivo and provides a measurement of changes in intracellular Ca2+. The mechanism involves a conformational change in myosin light chain kinase on binding of Ca2+–calmodulin, which leads to a change in the FRET signal from the engineered biosensor. Wang et al. (2013) concluded that in chronically AII-infused mice there is an elevation in [Ca2+]i in femoral artery smooth muscle. This increase in Ca2+ (∼80 nm) causes an active vasoconstriction and increased vascular tone. While a small component of this could be explained by a local effect of AII (∼9%) on the AII type 1 receptor, by far the greater contribution came from activation of the SNS, as concluded from the inhibitory effects of hexamethonium (a preganglionic blocker) and prazosin (an α1-adrenoceptor inhibitor). These results are therefore consistent with studies suggesting that AII exerts effects centrally on the SNS. In this scenario, AII is envisaged to affect areas of the brain outside of the blood–brain barrier, perhaps through secondary intermediates, such as reactive oxygen species. Wang et al. (2013) further concluded that chronic AII infusion did not lead to any observable alterations in vascular reactivity or artery remodelling. As the authors indicate, however, their measurements of diameter are restricted to an external diameter and this may not truly reflect intraluminal diameter because ‘folding’ of the wall would be expected during contraction. That being acknowledged, only relatively small changes in diameter were being studied, which lessens this concern. It could be argued that this study did not produce hypertension, per se, because an increase in mean arterial blood pressure of only some 8 mmHg was reported. As discussed by the authors, this may relate to blood pressure measurements being made under anaesthesia. Alternatively, it could perhaps be argued that the results are all the more impressive and that involvement of the SNS in a low-dose AII infusion (400 ng kg−1 min−1) model is more convincing than in higher-dose models, where multiple mechanisms may be activated simultaneously. An interesting question is, how do these in vivo results relate to recent studies indicating that prolonged exposure to vasoconstriction induces a remodelling that is characterized by inward eutrophic remodelling (Martinez-Lemus et al. 2009), often within the time frame used in the study of Wang et al. (2013)? As mentioned earlier, it is conceivable, as discussed by Wang et al. (2013), that restricting measurements to external diameters did not give a true indication of the effects of constrictor agents on luminal diameter. Furthermore, it could be questioned whether a true passive diameter was obtained in vivo with superfusion of 0 mm Ca2+ buffer. In addition, their approach cannot distinguish relative changes occurring within the matrix and cellular components of the vessel wall. Future studies of passive dimensions and mechanics performed in vitro, following conduct of the in vivo studies, could address this issue. Obviously, femoral arteries are not generally considered as resistance vessels, although it is interesting to note the substantial level of tone exhibited by these vessels (33% in control mice). While the authors argued that the femoral artery would be exposed to the same circulating factors and neural activity, it is likely that vascular heterogeneity could lead to differing vascular responses, given that different vascular beds demonstrate variation in ion channel and receptor populations. Furthermore, the femoral artery would be likely to differ in responses to alterations in haemodynamics; for example, myogenic reactivity is expressed more in smaller arteries and arterioles. Interestingly, in another study the authors found a different diameter response in femoral arteries compared with small cremaster muscle arterioles when hexamethonium was administered intraperitoneally (Mauban et al. 2013). These differing responses perhaps indicate a difference in innervation or complex responses secondarily to local changes in haemodynamics. Some of the unavoidable limitations of the study were the single time point study design, the requirement for anaesthesia and, perhaps, the use of the femoral artery to study what could be argued to be a resistance vessel phenomenon. On the positive side, this approach can be used for the study of many vascular beds in in vivo conditions. The utility of the technique is increased from the authors’ previous studies, because they now provide an approach for calibrating the in situ Ca2+ measurements. Overall, the study of Wang et al. (2013) provides two important advances. Does it solve the cellular basis for hypertension? Almost certainly not, given the probable multifactorial basis of this disorder and the temporal aspects of its development and maintenance. It does, however, demonstrate that in the conditions of these experiments, the increased vascular resistance in response to AII infusion can be mediated via the SNS. Furthermore, the in vivo FRET technique for measuring changes in intracellular Ca2+ provides an exciting methodological advance that can potentially be used in many studies of vascular reactivity in in vivo conditions.