All of the components required for angiotensin II formation are expressed locally in human atherosclerotic lesions, including a long suspected player cathepsin G

Without question, the renin–angiotensin system is recognized as one of the major blood pressure- regulatory systems. The renin–angiotensin system begins with the cleavage of a 50-kDa plasma glycoprotein, angiotensinogen, at the amino-terminal end of the protein by renin to give the biologically inactive decapeptide angiotensin I. Angiotensin I is then converted to the biologically active octapeptide angiotensin II by the catalase angiotensin-converting enzyme (ACE). However, in the last decade, many alternative pathways of angiotensin II formation have been identified, and much attention has focused on angiotensin II production by a group of serine proteinases synthesized and released by activated leukocytes. Of these serine proteinases, human heart chymase, which is expressed by cardiac mast cells, mesenchymal interstitial cells and endothelial cells, has garnished much attention as being a significant replacement of ACE [1]. Nonetheless, several other enzymes [2–4], most notably cathepsin G [5], have also been shown to produce angiotensin II from angiotensin I in vitro but, to date, the physiologic significance of these additional angiotensin II- generating pathways has not been elucidated in vivo. A participatory role for locally formed angiotensin II is considered in the pathology of hypertensive heart disease, congestive heart failure and acute myocardial infarction. This additional role of angiotensin II has been demonstrated in both primary and secondary prevention trials where pharmacological ACE inhibition improves the morbidity and mortality rates of patients with various cardiovascular diseases [6,7]. However, unlike ACE [8,9] and chymase [9], direct evidence from large groups of patients is lacking with respect to the physical presence of cathepsin G in human atherosclerotic lesions. Furthermore, despite the consistent evidence provided from studies performed in vitro [10–13] on the prognostic role of cathepsin G as a legitimate generator of angiotensin II, the evidence available in vivo linking lesion-specific cathepsin G expression and elevated tissue angiotensin II levels, akin to that shown for ACE and chymase, has also been lacking. In the present issue of the journal, Legedz et al. [14] offer much needed physical evidence linking cathepsin G to human vascular tissue, where it has the potential to contribute to local elevations in angiotensin II production at the site of human atherosclerotic lesions. High levels of angiotensin II have been demonstrated in experimental and human vascular lesions post vascular injury (neointimal formation) [15,16] or as the result of atherosclerosis [8]. In a study by Diet et al. [8], ACE, angiotensin II, and its receptor, have been shown to colocalize in areas of inflammation in human atherosclerotic lesions. In both early and intermediate-stage atherosclerotic lesions, immunoreactive angiotensin II was detected prominently in regions of fat-laden macrophages and in association with T lymphocytes. In advanced lesions, angiotensin II immunoreactivity was also localized to the endothelium of the microvasculature throughout the plaques, thus providing a direct physical link between endogenous angiotensin II and lesion development at all stages. In another example of angiotensin II participation in vascular disease, chronic administration of exogenous angiotensin II was shown to promote atherosclerotic lesion formation [17,18] and aneurysms [17] in a prominent atherosclerosis mouse model, the apolipoprotein E-deficient mouse. The numerous discoveries of the actions of angiotensin II at the level of the vascular tissue have revolutionized our thinking of the role of this octapeptide in cardiovascular disease. Evidence indicates that angiotensin II is more than simply a hormone that exerts haemodynamic and renal actions because it can have local, biologically active effects on endothelial and smooth muscle cells and on processes that regulate immunity and inflammation. Specifically, angiotensin II is a major mediator of oxidative stress as it has been shown to activate membrane NADH and NADPH oxidase activity in cultured vascular smooth muscle cells, resulting in the production of superoxide anion and, subsequently, hydrogen peroxide [19], which are both involved in intracellular second messenger signaling pathways [20]. Angiotensin II stimulated macrophage NADPH oxidase may also contribute to the modification of intimal-trapped lipoproteins [21,22]. Subsequent uptake of these modified lipoproteins by resident macrophages, via a number of scavenger–receptor-mediated pathways, has been shown repeatedly to cause the generation of macrophage-derived foam cells [23], which are present in all stages of human atherosclerosis. Angiotensin II has been shown to induce monocyte chemotactic protein-1 mRNA expression in monocytes and vascular smooth muscle cells [24], an action that can result in the recruitment of more inflammatory cells to the site of the developing lesion. Angiotensin II induces endothelial dysfunction and activates the expression of the proinflammatory phenotype of human vascular smooth muscle cells [25]. Through pleiotropic activation of nuclear factor-κB transcription factors, angiotensin II has been shown in vitro to induce the synthesis of both interleuken-6 [26] and tumour necrosis factor-α [27]. Collectively, all of these tissue-specific actions of angiotensin II demonstrate that the protein can play a key role in the initiation and amplification of pathobiological events that lead to vascular disease. Furthermore, the action of angiotensin II on the vessel wall should not be viewed as occurring in isolation, but most likely as operating synergistically with other cardiovascular risk factors, such as dyslipidemia and diabetes, further enhancing the atherogenic process. Cathepsin G is a neutral serine proteinase, which was originally found to be expressed and synthesized at the promyelocyte stage of development and to be packaged in the azurophil (primary) granules of leukocytes [28,29]. The amino acid composition and crystal structure of cathepsin G are both known [30–32]. Furthermore, cathepsin G has chymotrypsin-like specificity, preferring to cleave at Phe in the P1 position [33]. Cathepsin G has a number of potential substrates and activities that give it a potentially broad biologically significant role [34], although it is not clear if any of these potential properties of the enzyme have any physiological relevance. One unique property of cathepsin G is its ability to catalyse the conversion of angiotensin I to angiotensin II [10–12]. In vitro, conversion of angiotensin I to angiotensin II is one of the fastest reactions rates known for cathepsin G, giving it biological significance [12]. Cathepsin G has also been shown to generate angiotensin II directly from angiotensinogen [10], which is a property shared neither by ACE nor chymase [35]. As mentioned above, cathepsin G is localized primarily in the azurophil granules of leukocytes and is released in response to physiological stimuli [36]. However, cathepsin G has since been shown to be expressed on the surface of both human neutrophils and monocytes [13,36,37]. Furthermore, this membrane-bound cathepsin G is catalytically active and the extracellular expression of this protein by leukocytes is markedly up-regulated by signals that are relevant to infection and inflammation [13,36]. Both of these latter properties are important if cathepsin G is to have a relevant role in the process of atherogenesis given that the hypothesized site of angiotensin II formation would be extracellular, and also that a pro-inflammatory state of the vessel wall is more conducive to promoting the development of atherosclerotic lesions. Using complementary approaches of semi-quantitative reverse transcriptase-polymerase chain reaction, in situ hybridization and immunohistochemistry, Legedz et al. [14] examined atheroma plaques (type IV–V lesions), fatty streaks (type II–III lesions) and macroscopically intact tissue (type I lesions) obtained during carotid endarterectomy in 21 hypertensive patients, for the presence of renin, angiotensinogen, ACE, cathepsin G and D, and the angiotensin type 1 and 2 receptors. In their study, only renin and type 2 receptors could not be detected. Compared to intact tissue, ACE and cathepsin G mRNA expression were increased five-fold and two-fold, respectively, in more advanced (type IV-V) lesions, but not in early stage (type II–III) lesions, suggesting that both of these enzymes would have a greater impact on later stages of atherosclerotic lesion development. By contrast to the noted increase in the expression of ACE and cathepsin G, angiotensinogen mRNA levels did not change significantly as the atherosclerotic lesions matured. This finding suggests that a concomitant increase in angiotensin II production accompanying the increase in expression of both ACE and cathepsin G could only occur if the basal level of locally derived angiotensinogen substrate pool was significantly large enough to compensate for an increase in consumption as angiotensin II production increases, or that non-locally produced angiotensinogen is accessible to this substrate pool. Unfortunately, angiotensin II levels in these tissues were not measured so we are unable to confirm if angiotensin II levels did rise as would have been expected. Further tissue analysis also revealed that, compared to intact tissue, type 1 receptor expression decreased by 2.5-fold and 1.4-fold in atheroma and fatty streak lesions, respectively. This was as expected because an increase in angiotensin II production would cause type I receptor-expression to be down-regulated. The noted absence of measurable levels of renin would suggest that conversion of angiotensinogen to angiotensin I would most likely be mediated by cathepsin G because ACE cannot catalyse this first step, with further conversion of angiotensin I to angiotensin II being carried out by both cathepsin G and ACE. Certainly, other enzymes, namely chymase [38], elastase and proteinase 3 [39], could also facilitate the conversion of angiotensin I to angiotensin II and, although the presence of these three serine proteinases was not measured by Legedz et al. [14], their participatory role should not be dismissed. Regardless of the true number of different serine proteinases present, Legedz et al. [14] have provided substantial physical evidence that, in the absence of renin, all components required for angiotensin II formation from angiotensinogen are expressed locally in human atherosclerotic lesions. Furthermore, their current findings suggest that, in the absence of renin, cathepsin G could play a significant role as the major angiotensin I-generating enzyme. The enhanced expression of both ACE and cathepsin G permit for a concomitant increase in local angiotensin II production within human atherosclerotic lesions giving further credence to the participation of angiotensin II in the modulation of the disease. In conclusion, the identification of cathepsin G within human atherosclerotic lesions and the significant positive correlation between angiotensinogen, cathepsin G and ACE support the body of experimental evidence indicating that locally generated angiotensin II plays an important role in the progression of vascular disease, and that restoring the local balance of these mediators could constitute an important therapeutic goal. As a therapeutic strategy, inhibiting tissue ACE appears to be an effective target for preventing premature death, myocardial infarction, and stroke in patients who are at high risk for vascular disease. Would a similar regulation of cathepsin G activity also prove to be therapeutically beneficial? Before this question can be answered, further experimental evidence regarding the participatory role of cathepsin G in animal models of atherosclerosis is warranted. However, the evidence is mounting regarding alternative angiotensin II- generating pathways that involve locally produced serine proteinases. Future animal studies performed in vivo, and based on the findings of Legedz et al. [14], should allow us to gain not only a better understanding of the role of these alternate systems in cardiovascular pathobiology, but also a better understanding of the disease process at the level of the vascular wall. Acknowledgement S.C. Whitman is the recipient of a Great-West Life & London Life New Investigator award from the Heart and Stroke Foundation of Canada and is supported by the Heart and Stroke Foundation of Ontario Grant NA-5086 and Canadian Institutes of Health Research Grants MOP-53344 and GHS-60663. The author apologizes to those investigators whose excellent contributions to the field of angiotensin II, cathepsin G and hypertension research could not be included in this commentary due to space limitations.